HK1208037B - Expression sequences - Google Patents

Description

expressed sequence

The present invention relates to regulatory elements of the Pichia pastoris (P. pastoris) expression system and their use in a method for producing a protein of interest (POI).

Background Art

Protein secretion has been successfully achieved in both prokaryotic and eukaryotic hosts. The most prominent examples are bacteria such as Escherichia coli, yeasts such as Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, filamentous fungi such as Aspergillus awamori or Trichoderma reesei, or mammalian cells such as CHO cells. Although some proteins can be easily secreted with high efficiency, many other proteins are only secreted at relatively low levels.

Heterologous gene expression in a host organism requires a vector that allows for stable transformation of the host organism. This vector or expression vector must provide a functional promoter for the gene, located near the 5' end of the coding sequence. Transcription is thus regulated and initiated by this promoter sequence.

The secretory pathway typically begins with the translocation of transmembrane polypeptides and polypeptides intended for secretion into the lumen of the endoplasmic reticulum (ER). To this end, these proteins possess an amino-terminal preceding sequence, also known as a "leader sequence," which contains or consists of a signal peptide and an optional secretory pro-peptide. Signal peptides typically consist of 13-36 relatively hydrophobic amino acids. Signal peptides have a common structure: a short, positively charged amino-terminal region (n-region); a central, hydrophobic region (h-region); and a highly polar carboxy-terminal region (c-region), which contains a cleavage site for signal peptidase. On the lumenal side of the ER, the signal peptide is removed by a signal peptidase. After successful folding of the nascent polypeptide by ER chaperones and foldases, the protein is further directed out of the ER. This process is supported by the presence of an N-terminal pro-sequence, for example, in the precursor of the Saccharomyces cerevisiae mating factor α (MFα). The protein is then transported to the Golgi network and ultimately to the plasma membrane for secretion into the supernatant. The leader peptide is (presumably) cleaved from the protein by proteases within the Golgi, such as the Kex2 protease of Saccharomyces cerevisiae.

Because most yeasts do not secrete significant amounts of endogenous proteins and their extracellular proteomes are not extensively characterized, the number of secretion sequences available for yeast is limited. Therefore, fusions of the target protein with the mating factor α leader peptide (MFα) of Saccharomyces cerevisiae are used to drive secretory expression in a variety of yeast species, including Pichia pastoris, Kluyveromyces, and Zygosaccharomyces. Unfortunately, proteolytic processing of MFα by Kex2 protease often results in heterologous N-terminal amino acid residues in the product.

EP 324 274 B1 describes improved expression and secretion of heterologous proteins in yeast using a truncated Saccharomyces cerevisiae alpha-factor leader sequence, and EP 301 669 B1 describes a Kluyveromyces alpha-factor leader sequence for secretion of heterologous proteins.

Alternatively, the signal peptides of Saccharomyces cerevisiae phosphatase (PHO5, DK3614), Saccharomyces cerevisiae sucrose invertase (SUC, WO84/01153), and yeast aspartic proteinase 3 (YAP3, EP792367B1) can be used for secretory expression in yeast. EP0438200 (A1) discloses the signal peptide sequence of Saccharomyces cerevisiae SUC2 for expression in Pichia pastoris.

US5268273 describes a Pichia pastoris acid phosphatase (PHO1) signal sequence which is in most cases weaker than MFa.

US7741075 describes a Pichia pastoris PIR1 secretion signal peptide for recombinant protein expression, and Pichia pastoris PIR1 and PIR2 anchor domain peptides for recombinant surface display.

Khasa et al. (2011) (Yeast. 28(3): 213-26) described the isolation of the P. pastoris PIR gene and its application in cell surface display and recombinant protein secretion, especially the secretion of recombinant proteins in P. pastoris using the pre-pro signal of the PpPir1p protein, without comparison with MFα.

WO2011073367A1 and Kottmeier et al. 2011 (Applied Microbiology and Biotechnology. 91: 1, 133-141) describe a hydrophobin signal sequence that mediates efficient secretion of recombinant proteins in Pichia pastoris, in particular using the pre-sequence or pro-sequence of Trichoderma reesei hydrophobin for the secretion of eGFP in Pichia pastoris.

During the sequencing of the Pichia pastoris genome, 54 different sequences were identified as predicted signal peptides, which include a cleavage site to facilitate protein secretion (De Schutter et al. Nature Biotechnology doi: 10.1038/nbt.15442009).

US2011/0021378A1 describes an identified set of 54 Pichia pastoris genes that include a signal sequence, of which residues 1-21 of SEQ ID 8 are listed in US2011/0021378A1, and a cleavage site to provide for protein secretion.

EP2258855A1 describes regulatory sequences for an expression system derived from Pichia pastoris, in which the signal and leader peptide sequence of the Pichia pastoris Epx1 protein serves as a 57-amino acid leader sequence to facilitate expression and secretion of the target POI protein. The cleavage site for the signal peptidase is predicted to follow the signal sequence, between positions 20 and 21, indicated by a hyphen in the cleavage site sequence: VSA-AP (SEQ ID 6).

It would be desirable to provide alternative regulatory elements suitable for expressing POIs in recombinant eukaryotic host cells, as well as simple, efficient methods for producing secreted proteins in eukaryotic cells, preferably by producing a homologous N-terminus of the POI.

Summary of the Invention

This object is achieved by the presently claimed subject matter.

The present invention provides an isolated nucleic acid encoding a leader sequence selected from the group consisting of:

a) a leader peptide having the amino acid sequence of SEQ ID 10 or a functional variant thereof having one or two point mutations,

b) a leader peptide having an amino acid sequence selected from the group consisting of SEQ ID 11, 12, 13 and 14,

c) a signal peptide having the amino acid sequence of SEQ ID 1 or a functional variant thereof having one or two point mutations (preferably excluding SEQ ID 2), and

d) a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID 2, 3, 4 and 5 (preferably excluding SEQ ID 2).

The present invention further provides a leader sequence selected from:

a) a leader peptide having the amino acid sequence of SEQ ID 10 or a functional variant thereof having one or two point mutations,

b) a leader peptide having an amino acid sequence selected from the group consisting of SEQ ID 11, 12, 13 and 14,

c) a signal peptide having the amino acid sequence of SEQ ID 1 or a functional variant thereof having one or two point mutations (preferably excluding SEQ ID 2), and

d) a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID 2, 3, 4 and 5 (preferably excluding SEQ ID 2).

The specific leader peptide is thus characterized by a 36 amino acid (aa) leader sequence, of which the N-terminal 20 aa sequence is a specific signal peptide.

The specific signal peptide is therefore characterized by a 20 aa signal sequence, in particular excluding the C-terminal extension, for example a 21 aa sequence including a C-terminal extension by further amino acids such as alanine.

SEQ ID 1: MKSTNLILAIAAASVVSA, wherein

X at position 3 is F or L

X at position 16 is A or T

For example, EpxL-A, a 20 amino acid (aa) signal peptide, has the amino acid sequence of SEQ ID 1, wherein X at position 3 is L, and X at position 16 is A (SEQ ID 2).

According to a specific example, the EpxL-A, a 20 amino acid (aa) signal peptide, has the amino acid sequence of SEQ ID 1, wherein X at position 3 is F, and X at position 16 is A (SEQ ID 3).

Specifically, the present invention provides a signal peptide or a nucleic acid encoding the signal peptide, wherein the signal peptide has an amino acid sequence selected from the group consisting of SEQ ID 2, 3, 4 and 5.

SEQ ID 2: MKSTNLILAIAAASVVSA

SEQ ID 3: MKSTNLILAIAAASVVSA

SEQ ID 4: MKSTNLILAIAAASVVSA

SEQ ID 5: MKSTNLILAIAAASVVSA

Amino acid substitutions at positions 3 and 16 are indicated in bold and underlined.

Nucleic acid molecules encoding the signal peptides of the present invention are also referred to herein as "signal sequences."

The leader sequence defined above in the present invention particularly has the amino acid sequence of SEQ ID 10.

SEQ ID 10:

MKSTNLILAIAAASVVSAAPVAPAEEAANHLHKR, among which

X at position 3 is F or L

X at position 16 is A or T

Specifically, the present invention provides a leader sequence or a nucleic acid encoding the leader sequence, wherein the leader sequence has an amino acid sequence selected from the group consisting of SEQ ID 11, 12, 13 and 14.

SEQ ID 11: MKSTNLILAIAAASVVSAAPVAPAEEAANHLHKR

SEQ ID 12: MKSTNLILAIAAASVVSAAPVAPAEEAANHLHKR

SEQ ID 13: MKSTNLILAIAAASVVSAAPVAPAEEAANHLHKR

SEQ ID 14: MKSTNLILAIAAASVVSAAPVAPAEEAANHLHKR

Amino acid substitutions at positions 3 and 16 are indicated in bold and underlined.

For example, EpxL-KR, a 36 amino acid (aa) truncated leader sequence, has the amino acid sequence of SEQ ID 10, wherein X at position 3 is L, and X at position 16 is A.

According to a specific example, EpxL-KR, a 36 amino acid (aa) leader peptide, has the amino acid sequence of SEQ ID 10, wherein X at position 3 is F, and X at position 16 is A (SEQ ID 12).

The leader sequence of the present invention having the amino acid sequence of SEQ ID 10, 11, 12, 13 or 14, or a functional variant thereof having one or two point mutations, is also referred to herein as a "truncated leader sequence".

In particular, the nucleic acid encoding the truncated leader sequence comprises or consists of a nucleic acid encoding a signal peptide, wherein the signal peptide is selected from the group consisting of SEQ ID 2, 3, 4 and 5, preferably excluding SEQ ID 2. In particular, the nucleic acid encoding the truncated leader sequence comprises or consists of a nucleic acid encoding a leader peptide, wherein the leader peptide is selected from the group consisting of SEQ ID 11, 12, 13 and 14.

In particular, the truncated leader sequence comprises or consists of a signal peptide selected from the group consisting of SEQ ID 2, 3, 4 and 5, preferably excluding SEQ ID 2, or consists of a leader peptide selected from the group consisting of SEQ ID 11, 12, 13 and 14.

A particularly preferred nucleic acid encodes a signal peptide of the amino acid sequence of SEQ ID 3 or a leader peptide of SEQ ID 12; preferably, the nucleic acid sequence encoding the signal peptide or leader peptide is SEQ ID 16 or SEQ ID 19, respectively. A particularly preferred leader sequence is a signal peptide having the amino acid sequence of SEQ ID 3 or a leader peptide having the amino acid sequence of SEQ ID 12.

Therefore, one embodiment of the present invention is a leader sequence or a nucleic acid encoding such a leader sequence, wherein the leader sequence is

a) a signal peptide having the amino acid sequence of SEQ ID 3, preferably, wherein the encoding nucleic acid consists of the nucleotide sequence of SEQ ID 16 or a codon-optimized variant of SEQ ID 16; or

b) a leader peptide having or comprising the amino acid sequence of SEQ ID 12, or consisting thereof, preferably, wherein the encoding nucleic acid consists of the nucleotide sequence of SEQ ID 19 or a codon-optimized variant of SEQ ID 19.

Specifically, the nucleic acid of the present invention has

a) a nucleotide sequence encoding a signal peptide selected from the group consisting of SEQ ID 15, 16 and 17 (preferably excluding SEQ ID 15), or

b) a nucleotide sequence encoding a leader peptide selected from the group consisting of SEQ ID 18, 19 and 20, or

c) a nucleotide sequence which is a codon-optimized variant of SEQ ID 15, 16, 17, 18, 19 or 20.

A specific nucleic acid of the present invention encodes a leader sequence, which is a signal peptide of the amino acid sequence of SEQ ID 3. Preferably, the nucleic acid consists of the nucleic acid sequence of SEQ ID 16 or a codon-optimized variant of SEQ ID 16.

A specific nucleic acid of the present invention encodes a leader sequence, which is a leader peptide or a truncated leader sequence having the amino acid sequence of SEQ ID 12. Preferably, the nucleic acid consists of the nucleotide sequence of SEQ ID 19 or a codon-optimized variant of SEQ ID 19.

SEQ ID 15: Nucleotide sequence obtained from Pichia pastoris strain CBS7435 (CBS-KNAW Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands):

ATGAAG C TCTC C ACCAATTTGAT TC TAGCTATTGCAGCAGC T TCC G C C GTTGTCTCAGCT

SEQ ID 16: Nucleotide sequence used in Example 9, obtained from Pichia pastoris strain CBS7435, by PCR amplification using primers described in Example 9.

ATGAAGTTCTCTACCAATTTGATTCTAGCTATTGCAGCAGCTTCCGCCGTTGTCTCAGCT

SEQ ID 17: Nucleotide sequence obtained from Pichia pastoris strain DSMZ70382 (German Collection of Microorganisms and Cell Cultures)

ATGAAG T TCTC T ACCAATTTGAT CT TAGCTATTGCAGCAGC A TCC A C T GTTGTCTCAGCT

Nucleotides that differ in the above sequences are underlined.

SEQ ID 18: Nucleotide sequence obtained from Pichia pastoris strain CBS7435 (CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)

ATGAAGCTCTCCACCAATTTGATTCTAGCTATTGCAGCAGCTTCCGCCGTTGTCTCAGCTGCTCCAGTTGCTCCAGCCGAAGAGGCAGCAAACCACTTGCACAAGCGT

SEQ ID 19: Nucleotide sequence used in Example 5, obtained from Pichia pastoris strain CBS7435 by PCR amplification using primers described in Examples 1 and 5

ATGAAGTTCTCTACCAATTTGATTCTAGCTATTGCAGCAGCTTCCGCCGTTGTCTCAGCTGCTCCAGTTGCTCCAGCCGAAGAGGCAGCAAACCACTTGCACAAGCGT

SEQ ID 20: Nucleotide sequence obtained from Pichia pastoris strain DSMZ70382 (German Collection of Microorganisms and Cell Cultures)

ATGAAGTTCTCTACCAATTTGATCTTAGCTATTGCAGCAGCATCCACTGTTGTCTCAGCTGCTCCAGTTGCTCCAGCCGAAGAGGCAGCAAACCACTTGCACAAGCGT

In particular, the nucleic acid of the present invention is DNA.

In particular, the leader sequence of the present invention is a polypeptide.

According to a specific aspect, the present invention provides an isolated leader sequence, a truncated leader sequence, a signal peptide or a leader peptide, preferably the leader sequence has an amino acid sequence selected from the group consisting of SEQ ID 1, 2, 3, 4, 5, 10, 11, 12, 13 and 14, preferably excluding SEQ ID 2.

A particularly preferred leader sequence is a peptide consisting of the amino acid sequence of SEQ ID 3 or SEQ ID 12.

According to another aspect, the present invention further provides an expression component comprising a nucleic acid encoding a leader sequence operably linked to a nucleic acid sequence encoding a POI, wherein the leader sequence is selected from the group consisting of:

a) a leader peptide having the amino acid sequence of SEQ ID 10 or a functional variant thereof having one or two point mutations,

b) a leader peptide having an amino acid sequence selected from the group consisting of SEQ ID 11, 12, 13 and 14,

c) a signal peptide having the amino acid sequence of SEQ ID 1, or a functional variant thereof having one or two point mutations, and

d) a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID 2, 3, 4 and 5,

Preferably, fusions of a signal peptide consisting of the amino acid sequence of SEQ ID 2 and a polypeptide comprising (or consisting of) one or more immunoglobulin single variable domains, such as Nanobodies, are excluded therefrom.

However, according to a specific embodiment, the present invention provides a fusion of a leader peptide having an amino acid sequence selected from the group consisting of SEQ ID 11, 12, 13 and 14 and a POI, or provides a fusion of a signal peptide having an amino acid sequence selected from the group consisting of SEQ ID 2, 3, 4 and 5 and a POI, wherein the POI is selected from the group consisting of a growth factor, a hormone, a cytokine, an antibody and an antibody fragment, in particular, wherein the antibody or antibody fragment is selected from the group consisting of: scFv, miniantibody, bispecific antibody, trispecific antibody, tetraspecific antibody, Fab, Fc-fusion protein and full-length antibody such as IgG, IgA, IgD, IgM or Ig, preferably a full-length antibody, scFv or Fab, in particular including a fusion of a signal peptide consisting of the amino acid sequence of SEQ ID 2 and any of such antibodies or antibody fragments, in particular any of the full-length antibodies, scFv or Fab.

Particularly preferred nucleotide sequences encoding signal peptides or leader peptides for expression components consist of nucleotide sequences encoding the amino acid sequence of SEQ ID 3 or SEQ ID 12, preferably, SEQ ID 16 or SEQ ID 19, or codon-optimized variants of SEQ ID 16 or 19. Such signal peptides or leader peptides are preferably fused to any POI, including any antibody or antibody fragment, and particularly include fusions of a signal peptide or leader peptide consisting of the amino acid sequence of SEQ ID 3 or SEQ ID 12 and a polypeptide comprising or consisting of one or more immunoglobulin single variable domains, such as a nanobody.

According to another aspect, the present invention further provides an expression component comprising

a) a nucleotide sequence encoding a signal peptide selected from the group consisting of SEQ ID 15, 16 and 17, or

b) a nucleotide sequence encoding a leader peptide selected from the group consisting of SEQ ID 18, 19 and 20, or

c) a nucleotide sequence which is a codon-optimized variant of SEQ ID 15, 16, 17, 18, 19 or 20,

Preferably, fusion constructs formed by fusing the nucleotide sequence of SEQ ID 15 to a nucleotide sequence encoding one or more immunoglobulin single variable domains, such as Nanobodies, are excluded therefrom.

However, according to a specific embodiment, the present invention provides a fusion construct comprising the nucleotide sequence of SEQ ID 15 fused to a nucleotide sequence encoding an antibody or antibody fragment selected from the group consisting of: scFv, minibody, bispecific antibody, trispecific antibody, tetraspecific antibody, Fab, Fc-fusion protein and full-length antibody such as IgG, IgA, IgD, IgM or IgE, preferably a full-length antibody, scFv or Fab.

A particularly preferred nucleotide sequence is SEQ ID 16, encoding the signal peptide of SEQ ID 3.

Another particularly preferred nucleotide sequence is SEQ ID 19, encoding the leader peptide of SEQ ID 12.

Thus, according to a specific embodiment, the present invention provides a fusion construct comprising a nucleotide sequence of SEQ ID 16 or 19 fused to a nucleotide sequence encoding any protein of interest, in particular, the protein of interest is, for example, a POI as described herein, including but not limited to, any antibody or antibody fragment, in particular including fusion constructs, wherein the POI is a polypeptide comprising or consisting of one or more immunoglobulin single variable domains, such as a nanobody.

By means of the expression module according to the invention it is possible for the first time to provide the expression of a POI, in particular the expression of a secreted mature POI, with the correct, native N-terminal amino acid residue, in particular without an additional alanine at the N-terminus.

In particular, the POI is selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, process enzymes, growth factors, hormones and cytokines, or wherein the POI mediates the production of host cell metabolites, preferably selected from the group consisting of antibodies or fragments thereof, growth factors, hormones and cytokines.

According to a specific embodiment, the antibody or antibody fragment thereof is selected from the group consisting of: scFv, miniantibody, bispecific antibody, trispecific antibody, tetraspecific antibody, Fab, Fc-fusion protein and full-length antibody such as IgG, IgA, IgD, IgM or IgE, preferably a full-length antibody, scFv or Fab.

In particular, the expression component of the present invention is a fusion of a nucleic acid of the present invention and a nucleic acid encoding a POI, and is thus a non-naturally occurring nucleic acid. The present invention further provides a fusion protein of a leader sequence of the present invention and a POI, and is thus a non-naturally occurring fusion protein.

Therefore, the leader sequence is modified to improve the production of POI, for example, to increase secretion and improve quality, such as a correct N-terminus, and a leader sequence different from that of the prior art is used.

The expression component of the present invention particularly encodes a leader sequence consisting of a signal peptide or a signal peptide extended by a pro-sequence, wherein the pro-sequence consists of APVAPAEEAANHLHKR (SEQ ID 7), which is a part of the natural full-length leader sequence of the Pichia pastoris Epx1 protein, i.e., a 16 amino acid sequence identical to the 21st to 36th amino acid sequence of the 57 amino acid sequence of SEQ ID 8 (MKFSTNLILAIAAASTVVSAAPVAPAEEAANHLHKRAYYTDTTKTHTFTEVVTVYRT).

Compared to the prior art leader sequences such as SEQ ID 21, the two leader sequences of the present invention, the signal peptide and the truncated peptide, unexpectedly have unexpectedly improved properties, wherein,

SEQ ID 21: MKLSTNLILAIAAASAVVSAA,

According to US 2011/0021378 A1, it is also referred to herein as EpxL-AA (21 amino acids).

Furthermore, the present invention shows improved performance compared to the full-length leader sequence of the Pichia pastoris Epx1 protein described in EP2258855A1, which is the 57 amino acid sequence of SEQ ID 8 or a variant with leucine substituted at position 3 thereof.

This is all the more surprising since, according to the present invention, the 20 amino acid signal peptide without the original sequence or the truncated leader sequence are only 35% and 63% of the length of the full-length leader sequence, respectively.

The leader sequence of the present invention has specific advantages when fused to the POI compared to fusion with the alpha mating factor (αMF) leader sequence, such as increased secretion of the POI, and/or improved quality, such as a correct N-terminus, especially when the POI is a hormone, cytokine, antibody or antibody fragment, such as selected from the group consisting of: scFv, minibody, bispecific antibody, trispecific antibody, tetraspecific antibody, Fab, Fc-fusion protein and full-length antibody such as IgG, IgA, IgD, IgM or IgE, preferably a full-length antibody, scFv or Fab.

The expression cassette of the present invention explicitly excludes the nucleic acid encoding the native leader sequence (SEQ ID 8) of the Pichia pastoris Epx1 protein as described in EP2258855A1.

The expression component of the present invention further specifically excludes a nucleic acid encoding a leader sequence having amino acids 1 to 21 of the full-length leader sequence of Pichia pastoris Epx1 protein (ie, SEQ ID 11), as described in US 2011/0021378A1.

According to a particular aspect of the present invention, the expression component is optionally incorporated into an expression construct, such as a vector.

According to a further specific aspect of the present invention, the expression element or expression construct comprises a promoter operably linked to the nucleic acid encoding the leader sequence.

According to a specific embodiment of the present invention, the present invention provides a recombinant yeast host cell comprising the expression component of the present invention, particularly a yeast host cell line, more particularly a production cell line. According to a specific aspect of the present invention, the expression component is a vector, or a portion of a vector.

Preferably, the yeast is selected from the following species: Pichia, Candida, Torulopsis, Arxula, Hansenula, Ogatea, Yarrowia, Kluyveromyces, Schizosaccharomyces, Komagataella, preferably a methanol-trophic yeast, and particularly preferably Pichia pastoris, Komagataella pastoris, K. phaffii or K. pseudopastoris.

According to another embodiment, the present invention provides a method for producing a POI in a yeast host cell, comprising:

- providing a host cell of the present invention,

- cultivating the host cell to express the POI, and

- Purifying the POI to obtain a purified POI preparation.

According to the present invention, an expression module is preferably used to facilitate secretion of the recombinant gene in the yeast host cell, thereby increasing the yield of the secreted product.

Thus, the expression cassette of the present invention provides for efficient expression and secretion of a POI in a host cell transformed with said expression cassette. In this respect, the expression cassette of the present invention is understood to be a yeast expression cassette.

In particular, the POI is a recombinant protein, a term generally understood herein to include polypeptides and proteins, such as those produced by recombinant host cells. The POI can be a heterologous protein, for example, a protein heterologous to yeast, such as a protein obtained from a higher eukaryotic organism such as human, or a yeast species other than Pichia pastoris, or an artificial polypeptide or protein. Alternatively, the POI can be a homologous protein obtained from yeast, such as Pichia pastoris, that is not expressed or is expressed in insufficient amounts in native yeast that has not been transformed with the vectors of the present invention, but is expressed in desired amounts, such as overexpressed, in the recombinant yeast host cells of the present invention to produce a large amount of the POI or metabolite.

Specifically, the POI comprises an amino acid sequence having a native N-terminal amino acid sequence. The POI preferably comprises an amino acid sequence that does not include an additional alanine as an N-terminal amino acid residue. Specifically, the POI does not have an additional N-terminal amino acid residue derived from the leader sequence. This is crucial for the quality of the recombinant protein product and ease of production.

Specifically, the POI is a secreted polypeptide or protein, including a soluble extracellular molecule or a membrane-bound molecule.

Specifically, the POI is selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and vaccine-like proteins or particles, process enzymes, growth factors, hormones, and cytokines, or wherein the POI mediates the production of a host cell metabolite. The POI may also be an expression product that mediates the production of a host cell metabolite.

Specifically, the POI is selected from growth factors, hormones, cytokines, antibodies or antibody fragments, preferably selected from full-length antibodies, such as IgG, IgA, IgD, IgM or IgE, scFv, miniantibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, Fab, Fc-fusion proteins, preferably a full-length antibody, scFv or Fab.

Specifically, the POI includes an N-terminal amino acid residue other than alanine. Therefore, the VSA-AP (SEQ ID 6) signal peptidase cleavage site predicted in the prior art (EP2258855A1) is absent.

Specifically, the POI may be a secreted protein, for example, a mature protein, which may be an active form or a proform of the protein.

The method of the invention preferably provides for culturing the host cell in cell culture and obtaining the POI or metabolite, e.g. as a secreted POI, including membrane-bound or soluble or extracellular protein or metabolite, which is optionally purified from the cell culture supernatant.

According to a further aspect, the present invention provides the use of a nucleic acid or leader sequence according to the present invention, in particular a nucleic acid encoding a signal peptide or a truncated leader sequence according to the present invention, for secreting a POI from a host cell and/or increasing the secretion of a POI from a host cell, preferably wherein at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% of the secreted POI comprises a native N-terminal amino acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Promoter sequence pG1: SEQ ID 9.

Figure 2.1: Silver-stained SDS-PAGE of reduced supernatants of Pichia pastoris harboring EpxL-RT or MFα secreting pTRP, respectively.

Figure 2.2: Silver-stained SDS-PAGE of reduced supernatants of Pichia pastoris harboring EpxL-RT or MFα secreting eGFP, respectively.

Figure 2.3: Anti-HSA Western blot of reduced supernatants of Pichia pastoris secreting HSA harboring EpxL-RT or MFα, respectively.

Figure 3: Coomassie stained Western blot of HSA secreting Pichia pastoris supernatants with EpxL-RT for N-terminal sequencing.

Figure 4.1: Silver-stained SDS-PAGE of reduced supernatants of Pichia pastoris secreting pTRP harboring EpxL-RT, EpxL-KR or MFα, respectively.

Figure 4.2: Silver-stained SDS-PAGE of reduced supernatants of Pichia pastoris harboring EpxL-RT or MFα, respectively, secreting eGFP.

Figure 4.3: Supernatants of Pichia pastoris cells secreting HyHEL heavy chain or HyHEL light chain with EpxL-KR, respectively. HC: Western blot using anti-IgG γ chain antibody; LC: SDS-PAGE and silver staining.

Figure 5.1: Silver-stained SDS-PAGE of reduced supernatants of Pichia pastoris harboring EpxL-KR, EpxL-AA or EpxL-A, respectively, secreting eGFP.

Figure 5.2: Silver-stained SDS-PAGE of reduced supernatants of LC-secreting Pichia pastoris harboring EpxL-KR, EpxL-AA or EpxL-A, respectively.

Figure 5.3: Western blot of non-reduced supernatants of Pichia pastoris secreting HyHEL Fab under the control of pG1 with EpxL-A or MFα, respectively.

Figure 6: Western blot of supernatants of Pichia pastoris secreting human growth hormone (HGH), human somatotropin, interferon α2a, three different antibody fragments Fab1, Fab2, Fab3, and two different single-chain Fv antibody fragments scFv1 and scFv2, respectively, under the control of pG1 with EpxL-A.

DETAILED DESCRIPTION

Special terms have the following meanings throughout the specification.

As used herein, the term "cell line" refers to a clone constructed from a specific type of cell that has acquired the ability to proliferate over an extended period of time. The term "host cell line" refers to a cell line used to express an endogenous or recombinant gene or metabolic pathway product to produce a polypeptide or a cellular metabolite mediated by these polypeptides. A "production host cell line" or "production cell line" is generally understood to be a ready-to-use cell line that is cultured in a bioreactor to obtain a product in a production process, such as a POI. The term "yeast host" or "yeast cell line" or "yeast host cell" or "host cell" or "host" refers to any yeast cell that can be cultured to produce a POI or a host cell metabolite.

The term "expression" or "expression system" or "expression element" refers to a nucleic acid molecule comprising a desired coding sequence for an expression product, such as a POI, and a control sequence, such as a promoter, in operably linked form, such that a host transformed or transfected with these sequences is capable of producing the encoded protein or host cell metabolite. To effect transformation, the expression system may be contained on a vector; however, the relevant DNA may also be integrated into the host chromosome. Expression may involve secreted or non-secreted expression products, including polypeptides or metabolites. In particular, the term relates to a host cell and a compatible vector under appropriate conditions, for example, for expressing a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

As used herein, "expression construct" or "vector" is defined as the DNA sequence required for transcriptional cloning of a recombinant nucleotide sequence, i.e., the DNA sequence required for translation of the recombinant gene and its mRNA in a suitable host organism. An expression vector comprises an expression component and, additionally, typically comprises an origin or gene integration site for autonomous replication in the host cell, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring antibiotic resistance, such as zeocin, kanamycin, G418, or hygromycin), a plurality of restriction enzyme sites, a suitable promoter sequence, and a transcription terminator, which are operably linked together. As used herein, the terms "plasmid" and "vector" include both autonomously replicating and genomic integration nucleotide sequences.

Specifically, the term refers to a vehicle by which a DNA or RNA sequence (eg, a foreign gene) can be introduced into a host cell, thereby transforming the host and promoting expression (eg, transcription and translation) of the introduced sequence. Plasmids are preferred vectors of the present invention.

A vector typically contains DNA that is a transmissible agent into which foreign DNA can be inserted. A common method for inserting one DNA fragment into another DNA fragment involves the use of enzymes called restriction enzymes, which cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A "module" refers to a DNA coding sequence or DNA fragment that encodes an expression product that can be inserted into the restriction sites defined by the vector. The restriction sites of the module are designed to ensure that the module is inserted into the appropriate reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA and then carried by the vector into the host cell along with the transferable vector DNA. DNA fragments or sequences with inserted or added DNA, such as expression vectors, are also referred to as "DNA constructs." A common type of vector is a "plasmid," which is typically a self-contained double-stranded DNA molecule that can easily receive additional (foreign) DNA and can be easily introduced into a suitable host cell. Plasmid vectors typically contain coding DNA and promoter DNA and have one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a specific amino acid sequence for a particular polypeptide or protein, such as a POI. Promoter DNA is a DNA sequence that initiates, regulates, mediates, or controls the expression of coding DNA. Promoter DNA and coding DNA can be derived from the same gene or different genes and can be from the same or different organisms. Recombinant cloning vectors typically include one or more replication systems for cloning or expression, one or more markers for selection in a host, such as antibiotic resistance, and one or more expression modules.

As used herein, the term "functional variant" refers to variants having one or two point mutations in the amino acid sequence, for example, with respect to a regulatory sequence of the invention, such as a signal peptide having the amino acid sequence of SEQ ID 1, or with respect to a leader sequence having the amino acid sequence of SEQ ID 10, which have substantially the same signal or leader sequence activity as the unmodified sequence. Functional variants of the nucleic acids of the invention further include codon-optimized sequences, also referred to herein as "codon-optimized variants," which encode any signal peptide or leader sequence of the invention. Such codon optimization of nucleic acids is understood to be the systematic alteration of codons in a recombinant DNA to be expressed in a heterologous system to match the codon usage pattern used for expression in the organism. The present invention is particularly intended to increase the yield of the expressed protein.

It should be understood that the terms "signal peptide", "leader sequence" or "truncated leader sequence" as used herein always refer to the specific amino acids of SEQ ID 1 and SEQ ID 10, respectively, and also to their functional variants having one or two point mutations.

As used herein, the term "substantially the same signal or leader sequence activity" refers to an activity exhibited by a recombinant host cell that secretes substantially the same POI into the supernatant; for example, the level of POI in the supernatant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% of the level of POI in the supernatant provided by SEQ ID 1 or SEQ ID 10, respectively.

A point mutation is understood to be a modification of a polynucleotide to obtain the expression of an amino acid sequence that differs from the non-modified amino acid sequence, wherein the different amino acids are obtained by replacing or exchanging one or more single (discontinuous) amino acids. Preferred functional variants have one or more point mutations at positions 3 and 16 of SEQ ID 1 and SEQ ID 10, respectively.

Further preferred point mutations refer to the exchange of amino acids of the same polarity and/or charge. In this regard, amino acids refer to the 20 naturally occurring amino acids encoded by 64 triplet codons. These 20 amino acids can be divided into amino acids with neutral charge, positive charge and negative charge:

The " neutral " amino acids are shown below, along with their corresponding three-letter and one-letter codes, and polarity:

Alanine: (ALa, A) non-polar, neutral;

Asparagine: (Asn, N) polar, neutral;

Cysteine: (Cys, C) non-polar, neutral;

Glutamine: (Gln, Q) polar, neutral;

Glycine: (Gly, G) non-polar, neutral;

Isoleucine: (Ile, I) non-polar, neutral;

Leucine: (Leu, L) non-polar, neutral;

Methionine: (Met, M) non-polar, neutral;

Phenylalanine: (Phe, F) non-polar, neutral;

Proline: (Pro, P) non-polar, neutral;

Serine: (Ser, S) polar, neutral;

Threonine: (Thr, T) polar, neutral;

Tryptophan: (Trp, W) non-polar, neutral;

Tyrosine: (Tyr, Y) polar, neutral;

Valine: (Val, V) non-polar, neutral; and

Histidine: (His, H) polar, positive charge (10%) neutral (90%).

The " positively " charged amino acids are:

Arginine: (Arg, R) polarity, positive charge; and

Lysine: (Lys, K) polarity, positive charge.

The " negatively " charged amino acids are:

Aspartic acid: (Asp, D) polar, negative charge; and

Glutamic acid: (Glu, E) polarity, negative charge.

As used herein, the terms "isolated" or "isolated" with respect to nucleic acids, POIs, or other compounds refer to compounds that have been sufficiently separated from their naturally associated environment to be in a "substantially pure" form. The term "isolated" does not necessarily exclude artificial or synthetic mixtures in which other compounds or materials are present, or impurities that do not interfere with essential activity are present, and these may be present, for example, due to incomplete purification. Specifically, isolated nucleic acid molecules of the present invention are also meant to include those that have been chemically synthesized. The term "isolated nucleic acid" is sometimes used in reference to the nucleic acids of the present invention. This term, when applied to DNA, refers to a DNA molecule that has been separated from a sequence that is directly adjacent to the sequence in the naturally occurring genome of the source organism. For example, an "isolated nucleic acid" can include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or a DNA molecule integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Specifically, the term "isolated nucleic acid" as used herein does not include a nucleic acid encoding an EPX1 protein linked to a nucleic acid encoding a leader sequence of the present invention. When applied to RNA, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by isolated DNA as defined above. Alternatively, the term refers to an RNA molecule that has been substantially separated from other nucleic acids with which it is associated in its natural state (i.e., in a cell or tissue). "Isolated nucleic acid" (DNA or RNA) may further refer to a molecule that has been directly produced by biological or synthetic means and separated from other components present during its production.

As used herein, the term "leader sequence" should be understood as follows. The polynucleotide and nucleic acid coding regions in the expression assemblies of the present invention may be associated with additional coding regions encoding secretory or signal peptides to direct the secretion of the POI. Proteins destined for the secretory pathway have an N-terminal leader sequence that is cleaved from the mature protein once the initial protein chain is exported across the rough endoplasmic reticulum. The leader sequence induces the transport of the expressed protein toward or outside the cytoplasmic membrane, thereby facilitating the isolation and purification of the expressed protein. Typically, membrane proteins or secreted proteins that are transported to the periplasmic space, within the cell membrane, or outside the cell contain such an N-terminal sequence. Typically, after transport of the protein, the leader sequence is cleaved from the protein by specific cellular peptidases.

Proteins secreted by eukaryotic cells typically have a leader sequence fused to the N-terminus of the protein, which is cleaved from the intact or "full-length" protein to produce the secreted or "mature" form of the protein.

Specific examples of leader sequences of the present invention are a leader sequence consisting of a signal peptide, or a leader sequence consisting of a signal peptide and a prosequence, such as the truncated leader sequence described herein. A leader sequence consisting of the signal peptide of SEQ ID 1, or the truncated leader sequence of SEQ ID 10, is also referred to herein as a regulatory sequence.

As used herein, the term specifically refers to control sequences that regulate the expression of a POI. A leader sequence, also known as a leader peptide, is linked to the N-terminus of the POI amino acid sequence. A nucleic acid encoding the leader sequence is upstream and operably linked to the 5' terminus of the nucleic acid sequence encoding the POI. Any leader sequence of the present invention that is functionally active in the selected host cell can be used.

The term "native N-terminal amino acid residue" or "native N-terminal amino acid sequence" refers to one or more amino acids of an N-terminal sequence, e.g., the N-terminal amino acid residue of a POI, which is considered to be correct compared to the sequence of the POI to be expressed. Thus, the native N-terminal amino acid residue provides a native N-terminal or N-terminal region of the POI, which is a prerequisite for obtaining a correct complete amino acid sequence, e.g., obtaining a functional compound that does not contain any additional (redundant) N-terminal amino acid residues that are foreign to the POI. In general, any wild-type protein is understood to have a native N-terminal amino acid residue. In addition, recombinant proteins may also have a native N-terminal amino acid residue that is predefined and exhibits the desired protein properties.

In particular, when a signal peptide or truncated leader sequence of the present invention is directly linked to the native N-terminal amino acid residue of a POI, the released protein will contain, at least to some extent, a native N-terminal amino acid residue and will generally not contain an N-terminal extension of variable length. Preferred POI compositions are characterized by containing a correct N-terminal native N-terminal amino acid residue, at least to the extent that preferably predominantly the POI molecule, or at least 60, 65, 70, 75, 80, 85, 90, 95, 98 or 100% (w/w) of the POI molecule, without containing additional amino acid residues at the N-terminus, such as those derived from a signal peptide or prosequence or fragments thereof.

As used herein, the term "prosequence" refers to a precursor amino acid sequence that is operably linked to the N-terminus of a POI. The prosequence may also have a signal sequence operably linked to the N-terminus of the prosequence. Typically, the prosequence is cleaved from the POI to leave a mature form of the POI.

A specific example of a prosequence of the present invention is a portion of a truncated leader sequence having the following amino acid sequence:

SEQ ID 7: APVAPAEEAANHLHKR

That is, the original sequence: amino acids 21 to 36 of the truncated leader sequence of SEQ ID 10 were truncated.

As used herein, the term "operably linked" refers to a nucleotide sequence that is linked to a single nucleic acid molecule, such as a vector, in such a manner that the function of one or more nucleotide sequences is affected by at least one other nucleotide sequence present on the nucleic acid molecule. For example, a promoter is operably linked to a coding sequence of a recombinant gene when the promoter is capable of affecting the expression of the coding sequence. As a further example, a nucleic acid encoding a signal peptide is operably linked to a nucleic acid sequence encoding a POI when it is capable of expressing a secreted form of the protein, such as a proform of the mature protein or the mature protein. Specifically, such operably linked nucleic acids can be linked immediately, i.e., without further elements or nucleic acid sequences between the nucleic acid encoding the signal peptide and the nucleic acid encoding the POI.

As used herein, "promoter" refers to a DNA sequence that is capable of controlling the expression of a coding sequence or functional RNA. Promoter activity can be assessed by its transcriptional efficiency. This can be measured directly by measuring the amount of mRNA transcribed from the promoter, for example, by Northern hybridization, or indirectly by measuring the amount of the gene product expressed by the promoter.

As used herein, the term "protein of interest (POI)" refers to a polypeptide or protein produced in a host cell by recombinant technology, also referred to as a recombinant POI or a POI produced by a recombinant host cell. More specifically, a recombinant POI may be a protein that is not naturally produced in a host cell, i.e., a heterologous protein, or a protein that is present in the host cell itself, i.e., a host cell homologous protein, but which is produced by, for example, transformation with a self-replicating vector comprising a nucleic acid sequence encoding the POI, or by integrating one or more copies of a nucleic acid sequence encoding the POI into the host genome by recombinant technology, or by recombinantly modifying one or more regulatory sequences that control the expression of the gene encoding the POI, such as promoter or signal sequence modifications. In certain cases, a recombinant POI, whether heterologous or homologous, is overexpressed by a recombinant host cell to obtain a high yield of the product. In some cases, the term POI as used herein also refers to any metabolic product of the host cell mediated by the recombinantly expressed protein.

As used herein, the term "secretion" refers to the translocation of a polypeptide or protein, particularly a POI, through the cell membrane and cell wall of a host plant cell. A secreted POI may be part of the cell membrane, anchored in the cell wall as a membrane-bound protein, or released into the cell supernatant as a soluble protein.

It is to be understood that the term "secreted" as used herein in relation to a POI comprises inter alia the expression of the POI in its mature form (including the pro-form or active protein), as a membrane-bound POI or an extracellular POI.

As used herein, the term "recombinant" means "produced by or as a result of genetic engineering." Thus, a "recombinant microorganism" includes at least one "recombinant nucleic acid." Recombinant microorganisms include, in particular, expression vectors or cloning vectors, or are genetically engineered to contain recombinant nucleic acid sequences. A "recombinant protein" is produced by expressing the corresponding recombinant nucleic acid in a host.

The nucleic acid molecules or peptides/polypeptides/proteins of the present invention are preferably recombinant to provide a fusion of the leader sequence and the POI. As used herein, "recombinant" refers to the artificial joining of two originally separate sequence fragments, such as nucleic acid fragments separated by chemical synthesis or by genetic engineering techniques. "Recombinant" also includes reference to cells or expression components that have been modified by the introduction of heterologous nucleic acid, or to cells derived from such modified cells, but does not include changes in cells or vectors resulting from naturally occurring events (e.g., spontaneous mutations, natural transformation/transduction/transposition), such as those that occur without intentional human intervention.

The term "signal sequence" as used herein refers to a nucleic acid encoding a signal peptide, which is typically a short (3-60 amino acid length) peptide chain that guides protein transport. Signal peptides may also be referred to as targeting signals, transit peptides, or positioning signals. Signal peptides particularly include expressed proteins to be transported along the secretory pathway. Generally speaking, membrane proteins or secretory proteins transported to the periplasmic space, intracellular membranes, or extracellular space comprise this N-terminal signal sequence. Typically, once nascent protein chains are translocated to the endoplasmic reticulum, the signal peptide is excised from the mature protein by a signal peptidase.

A specific example of a signal peptide used in the present invention is the signal peptide of SEQ ID 1, or a functional variant thereof having one or two point mutations. The signal peptide used in the present invention generally has an inherent property that it can cleave the amino acid after alanine 20 even if the cleavage site of SEQ ID 6 is not present.

The signal peptide is encoded by a nucleic acid sequence that usually precedes the nucleic acid sequence encoding the POI, optionally with a prosequence downstream of the signal sequence and upstream of the POI coding sequence.

As used herein, the term "substantially pure" or "purified" refers to a preparation containing at least 50% (w/w), preferably at least 60%, 70%, 80%, 90% or 95% of a compound, such as a nucleic acid molecule or POI. The purity of the compound is measured by an appropriate method (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

During the identification and characterization of the newly isolated nucleic acid encoding the specific signal peptide of the present invention, it was surprisingly discovered that the signal peptide contains an intrinsic feature that cleaves the C-terminus independently of the following amino acid residues. This is also referred to herein as the "intrinsic secretion site" at the C-terminus. Therefore, once the leader sequence is cleaved, the amino acid sequence following the signal peptide sequence will have a correct, native N-terminal amino acid sequence.

Surprisingly, when using a nucleic acid encoding a signal peptide according to the invention, a cleavage site for a signal peptidase known in the art is located between the signal sequence and the prosequence of the native Pichia pastoris Epx1 leader sequence, i.e., between positions 20 and 21, as indicated by a hyphen in the sequence of the cleavage site: VSA-AP (SEQ ID 6), wherein the cleavage site can be omitted. Thus, an improved expression system can be provided for providing, at least to some extent, the correct, native N-terminal amino acid residue of the POI, which in particular does not include an additional alanine as the N-terminal amino acid residue, e.g., the majority (up to 100%) of the POI molecules comprise the native N-terminal sequence (as determined by LC-MS).

Thus, for the first time, an isolated nucleic acid encoding a 20-amino acid signal peptide is available ready-to-use. Prior art signal sequences (e.g., the signal sequence from US20110021378A1) always contain at least one additional amino acid residue at the C-terminus, which is an alanine, which can lead to an incorrect N-terminus of the protein to be expressed.

Likewise, it has been discovered that the truncated leader sequences of the present invention contain an intrinsic feature that cleaves the C-terminus independently of the following amino acid residues. This is also referred to herein as the "intrinsic cleavage site" at the C-terminus. Thus, once the leader sequence is cleaved, the amino acid sequence following the C-terminus of the truncated leader sequence or the C-terminus of the original sequence of the truncated leader sequence will have the correct native N-terminal amino acid sequence.

The isolated nucleic acid and expression vector of the present invention thus provide for the expression and secretion of a POI with a native N-terminal amino acid residue. This is all the more surprising because the secretion levels observed with a 20-amino acid signal peptide were much higher than those observed with a 21-amino acid sequence (i.e., with an additional alanine added to the C-terminus to extend the 20-amino acid signal peptide, as proposed in US20110021378A1).

Thus, another embodiment of the present invention is the use of a nucleic acid or leader peptide of the present invention for secreting a POI and/or increasing secretion of a POI from a host cell, preferably wherein after removal of the leader sequence of the present invention, at least 60, 65, 70, 75, 80, 85, 90, 95, 98, or 100% of the POI comprises a native N-terminal amino acid sequence. Preferably, secretion is increased by 1.15, 1.5, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more fold compared to the known signal peptide SEQ ID 21. Most preferably, the POI is an antibody or a fragment or derivative thereof, as further defined below.

Furthermore, it was unexpectedly discovered that a truncated leader sequence could be used, which is only 63% of the length of the existing natural full-length leader sequence and thus less than the effective portion of the full-length leader sequence of SEQ ID 8. Surprisingly, the use of the truncated leader sequence even increased the secretion amount compared to experiments performed using the full-length leader sequence.

According to the present invention, the isolated nucleic acid or expression assembly or vector may utilize other conventional regulatory elements or sequences that are naturally occurring or commonly used in recombinant expression systems, for example to provide constructs for high yield production of recombinant proteins. Examples of regulatory sequences include promoters, operators and enhancers, ribosome binding sites, and sequences that control initiation and termination of transcription and translation.

To demonstrate the functionality of the sequence of interest, an expression cassette or vector can be constructed to drive POI expression, and the amount of expression and/or secretion compared to constructs with traditional regulatory elements. Detailed descriptions of the experimental procedures are provided in the Examples below.

The identified sequences are amplified by PCR from Pichia pastoris using specific nucleotide primers, cloned into a yeast expression vector, functionally linked to the N-terminus of the POI or upstream of the POI sequence, and transformed into a yeast host cell line, such as Pichia pastoris, for high-level production of various secreted forms of the POI. To evaluate the effects of regulatory sequences (e.g., the signal sequence and truncated leader sequence of the present invention) on POI production, the yeast host cell lines obtained according to the present invention can be cultured in shake flask experiments and fed-batch or chemostat fermentations and compared with cell lines containing traditional regulatory elements.

By using the regulatory elements of the present invention, particularly sequences encoding signal peptides or truncated leader sequences, and expression vectors, the methods of the present invention preferably not only increase production by enhancing secretion, but also improve the quality of the POI in yeast host cells, particularly Pichia pastoris host cells. Increased POI secretion is determined based on the amount of POI secreted in the presence of the regulatory sequence, particularly the signal sequence or truncated leader sequence, compared to the existing elements, which increase protein secretion.

The POI can be a eukaryotic, prokaryotic, or synthetic polypeptide. It can be secreted as a mature protein, membrane-bound, or expressed extracellularly. The present invention also provides for the recombinant production of functionally equivalent variants, derivatives, and biologically active fragments of naturally occurring proteins. Functionally equivalent variants preferably possess substantially the same functional characteristics or activities.

The POI mentioned herein may be a product, which is homologous or heterologous to the eukaryotic host cell, preferably for therapeutic, prophylactic, diagnostic, analytical or industrial use.

The POI is preferably a heterologous recombinant polypeptide or protein produced by yeast cells.

In particular, the POI is a eukaryotic protein, preferably a mammalian protein.

The POI produced according to the present invention may be a multimeric protein, preferably a dimer or trimer.

According to one aspect of the present invention, the POI is a recombinant or heterologous protein, preferably selected from therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine and vaccine-like proteins or particles, process enzymes, growth factors, hormones and cytokines, or metabolites of the POI.

A particular POI is an antigen-binding molecule, such as an antibody or fragment thereof. Among the various specific POIs are antibodies, such as monoclonal antibodies (mAbs), immunoglobulins (Ig) or class G immunoglobulins (IgG), heavy chain antibodies (HcAbs), or fragments thereof, such as fragment-antigen binders (Fabs), Fds, single-chain variable fragments (scFvs), or engineered variants thereof, such as Fv dimers (diabodies), Fv trimers (trispecific antibodies), Fv tetramers, or minibodies and single-domain antibodies such as VHs, VHHs, or V-NARs.

Further specific antibodies are aprotinin, tissue factor pathway inhibitor or other protease inhibitors, as well as insulin or insulin precursors, insulin analogs, growth hormone, interleukins, tissue plasminogen activator, transforming growth factor a or b, glucagon, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GRPP, factor VII, factor VIII, factor XIII, platelet-derived growth factor 1, serum albumin, enzymes such as lipases or proteases, or functional homologs, functionally equivalent variants, derivatives and biologically active fragments having similar functions to the native protein. POIs can be structurally similar to the native protein and can be derived from the native protein by adding one or more amino acids to either or both of the C-terminus and N-terminus or to the side chains of the native protein, substituting one or more amino acids at one or more different sites in the native amino acid sequence, deleting one or more amino acids at one or both ends of the native protein or at one or more sites in the amino acid sequence, or inserting one or more amino acids at one or more sites in the native amino acid sequence. These modifications are known for the above-mentioned proteins.

The POI can also be selected from substrates, enzymes, inhibitors, or cofactors that provide a biochemical reaction within the host cell, with the goal of obtaining a product of the biochemical reaction or a cascade of reactions, for example, to obtain a host cell metabolite. Exemplary products are vitamins, such as vitamin B2, organic acids, and alcohols, the production of which can be increased after expression of the recombinant protein or POI of the present invention.

Specifically, the host cell for expressing the recombinant product of the present invention can be any yeast cell suitable for POI recombinant expression.

Preferred host cells are selected from the group consisting of Pichia, Candida, Torulopsis, Arxula, Hansenula, Ogatea, Yarrowia, Kluyveromyces, Saccharomyces or Komagataella, preferably a methylotrophic yeast, and particularly preferred are Pichia pastoris, Komagataella pastoris, K. phaffii or K. pseudopastoris.

Examples of preferred host cells of the present invention include, but are not limited to, Pichia, such as Pichia pastoris or Pichia methanolica, or Komagataella, such as K. pastoris, K. pseudopastoris, or K. phaffii.

More recent literature has divided and renamed Pichia pastoris as Komagataella pastoris, Komagataella phaffii, and Komagataella pseudopastoris. Pichia pastoris is used here as a synonym for all species, including Komagataella pastoris, Komagataella phaffii, and Komagataellapseudopastoris.

Examples of Pichia pastoris strains include CBS 704 (=NRRL Y-1603=DSMZ 70382), CBS 2612 (=NRRL Y-7556), CBS 7435 (=NRRL Y-11430), CBS 9173-9189 (CBS strains: CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands), and DSMZ 70877 (German Collection of Micro-organisms and Cell Cultures), and also cell strains available from Invitrogen, such as X-33, GS115, KM71, and SMD1168. All of the above cell strains have been successfully used for the production of transformants and for the expression of heterologous genes.

According to the present invention, preferably a yeast host cell is provided which is transformed with a vector comprising a promoter sequence operably linked to a signal sequence or truncated leader sequence of the present invention.

According to a preferred embodiment of the present invention, the expression component of the present invention comprises a promoter, such as the P. pastoris pGAP (glyceraldehyde phosphate dehydrogenase) promoter, the pAOX (alcohol oxidase) promoter, or SEQ ID 9 (Figure 1, pG1 promoter), or a functional variant thereof. Effective sequence activity can be achieved using only a portion of these promoter sequences. Specifically, the preferred partial promoter sequence consists of at least 150 consecutive bases or bp, more preferably, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, or at least 900 bp. Preferably, the 3' end of the promoter region is contained within the preferred partial promoter sequence.

In a preferred expression system, the promoter is an inducible or constitutive promoter. The promoter can be endogenous to the host cell or heterologous to the host cell. A preferred example of an inducible promoter is the pG1 promoter, which is induced under glucose-limiting conditions and has the nucleotide sequence of SEQ ID 9.

A particular host cell of the present invention comprises a heterologous or recombinant promoter sequence, which can be obtained from a cell strain different from the production host, for example, from another yeast strain, such as a strain of Saccharomyces cerevisiae. In another specific embodiment, a host cell of the present invention comprises a recombinant expression construct of the present invention comprising a promoter from a cell of the same genus, species, or strain as the host cell.

The promoter may be any DNA sequence that exhibits transcriptional activity in the host cell and may be obtained from genes encoding proteins homologous or heterologous to the host cell. The promoter is preferably obtained from genes encoding proteins homologous to the host cell.

For example, a promoter of the present invention can be obtained from yeast, such as a strain of Saccharomyces cerevisiae. However, a particularly preferred embodiment utilizes a promoter derived from Pichia pastoris for use in methods for producing a recombinant POI in a Pichia pastoris producer host cell line. The homology of the nucleotide sequence facilitates incorporation into host cells of the same genus or species, thereby enabling stable production of the POI and potentially increasing yields in industrial manufacturing processes. Furthermore, functionally active variants of promoters from other suitable yeasts, other fungi, or other organisms, such as vertebrates or plants, can be used.

Further suitable promoter sequences for yeast host cells may include, but are not limited to, promoters obtained from genes encoding metabolic enzymes known to be present in high concentrations in cells, for example, glycolytic enzymes such as triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol oxidase (AOX), lactase (LAC), and galactosidase (GAL).

Preferred examples of suitable promoters are yeast promoters, which comprise a DNA sequence as a promoter for gene transcription in yeast cells. Preferred examples are the Saccharomyces cerevisiae Mal, TPI, CUP, ADH or PGK promoters, or the Pichia pastoris glucose-6-phosphate isomerase promoter (PPGI), 3-phosphoglycerate kinase promoter (PPGK) or glyceraldehyde phosphate dehydrogenase promoter PGAP, alcohol oxidase promoter (PAOX), formaldehyde dehydrogenase promoter (PFLD), isocitrate lyase promoter (PICL), translation elongation factor promoter (PTEF), and Pichia pastoris enolase 1 (PENO1), phosphoglyceraldehyde isomerase (PTPI), α-ketoisocaproate decarboxylase (PTHI), ribosomal subunit protein (PRPS2, PRPS7, PRPS31, P The mitochondrial proteins involved in the synthesis of phosphodiesterases (PEPSIs), phosphoglycerate mutase (PGPM1), phosphotransferase (PTKL1), phosphatidylinositol synthase (PPIS1), iron-O2-oxidoreductase (PFET3), high-affinity iron permease (PFTR1), repressible alkaline phosphatase (PPHO8), N-myristoyltransferase (PNMT1), pheromone-responsive transcription factor (PMCM1), ubiquitin (PUBI4), single-stranded DNA endonuclease (PRAD2), and the major ADP/ATP carrier of the inner mitochondrial membrane (PPET9) were also detected.

If the POI is a protein homologous to the host cell, i.e., a protein naturally produced in the host cell, the expression of the POI in the cell can be regulated by exchanging its native promoter sequence with a promoter sequence that is heterologous to the host cell or homologous to the host cell but different from the native promoter sequence of the POI.

The purpose of introducing a new promoter can be achieved, for example, by transforming a host cell with a recombinant DNA molecule containing homologous sequences to the gene of interest to allow site-specific recombination, a promoter sequence, and a selectable marker appropriate to the host cell. Site-specific recombination can be generated to operably link the nucleotide sequence encoding the POI to the promoter sequence. This results in expression of the POI from a heterologous promoter sequence rather than from the native promoter sequence.

In a particularly preferred embodiment of the present invention, the promoter sequence of the present invention has an increased promoter activity compared to the native promoter sequence of POI.

According to the present invention, a wildcard vector or expression module of the present invention is also provided, comprising a signal sequence or truncated leader sequence of the present invention. This wildcard vector or expression module is readily available for binding to a target gene encoding a POI. Thus, the wildcard cell line is a pre-established host cell line characterized by its expression capacity. This embodies an innovative "universal" platform strategy for generating producer cell lines for producing POIs, for example, using site-specific module integration or site-specific recombinase-mediated module exchange. This novel host cell facilitates cloning of a target gene (GOI) into, for example, a predetermined genomic expression site to obtain a reproducible, efficient producer cell line.

According to a preferred embodiment, the expression vector of the present invention is a plasmid suitable for integration into the host cell genome in a single copy or multiple copies per cell. The recombinant nucleotide sequence encoding the POI can also be provided on an autonomously replicating plasmid in a single copy or multiple copies per cell. Preferred plasmids are eukaryotic expression vectors, preferably yeast expression vectors.

Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, and specially designed plasmids. Preferred expression vectors for use in the present invention can be any expression vector suitable for expressing recombinant genes in host cells and are selected based on the host organism. Recombinant expression vectors can be any vector capable of replicating or integrating into the genome of the host organism, also referred to as host vectors, such as yeast vectors, which carry the DNA construct of the present invention. A preferred yeast expression vector is suitable for expression in methanol-trophic yeasts, represented by the genera Hansenula, Ogatea, Pichia, Candida, and Torulopsis.

In the present invention, a plasmid obtained from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis, or pPUZZLE is preferably used as a vector.

According to a preferred embodiment of the present invention, recombinant constructs are obtained by binding the relevant genes to vectors. These genes can be stably integrated into the host cell genome by transforming host cells with such vectors. Recombinant cell lines can be used to produce polypeptides encoded by these genes by culturing the transformants in suitable culture medium. The expressed POI can then be isolated from the culture and purified using methods appropriate for the expression product, particularly to separate the POI from contaminating proteins.

The DNA sequence encoding the POI may also be operably linked to a suitable terminator sequence, such as the AOX1 (alcohol oxidase) terminator, the CYC1 (cytochrome c) terminator, or the TEF (translation elongation factor) terminator.

Expression vectors can include one or more phenotypic selectable markers, such as genes encoding proteins that confer antibiotic resistance or genes that provide an auxotrophic requirement. Yeast vectors typically contain an origin of replication from a yeast plasmid, an autonomously replicating sequence (ARS), or a sequence for integration into the host genome, a promoter region, sequences for polyadenylation, sequences for transcription terminators, and a selectable marker.

Methods for ligating DNA sequences, for example DNA sequences encoding a leader sequence and/or POI, a promoter and a terminator, respectively, and for inserting them into a suitable vector containing the necessary information for integration or replication in a host are known to the person skilled in the art and are described, for example, in "J. Sambrook et al., "Molecular Cloning 2nd ed.", Cold Spring Harbor Laboratory Press (1989)".

It will be appreciated that the vectors of the present invention can be constructed by first preparing a DNA construct containing the entire DNA sequence of the desired vector elements and inserting this construct into a suitable expression vector; or by sequentially inserting DNA fragments containing the genetic information for each element (e.g., signal, leader, or POI) and then ligating them. Alternatively, the individual elements of the expression component can be fused by PCR.

The present invention also employs polyclonal vectors, which have multiple cloning sites into which desired genes can be incorporated to provide an expression vector. In the expression vector, a promoter is placed upstream of the leader sequence and the POI gene and regulates the expression of these genes. In the case of a polyclonal vector, the promoter is placed upstream of the multiple cloning site because the leader sequence and POI gene are introduced into the multiple cloning site. The leader sequence gene can be fused to the POI gene via PCR or synthetic preparation, or the leader sequence gene can be provided in a vector or expression vector, and the POI gene can be introduced using standard cloning methods.

Expression vectors or components and DNA constructs provided by the present invention can be synthesized by established standard methods, such as the phosphoramidite method. The DNA construct can also be genomic or cDNA derived, such as by preparing a genomic or cDNA library, and by hybridization screening for a DNA sequence encoding all or part of the polypeptide of the present invention, using synthetic oligonucleotide probes, and using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989). Finally, the DNA construct can be a mixture of synthetic and genomic origins, a mixture of synthetic and cDNA origins, or a mixture of genomic and cDNA origins, using standard techniques by annealing synthetic, genomic or cDNA derived fragments, each of which corresponds to the various parts of the entire DNA construct, as appropriate.

It will be apparent to those skilled in the art based on state of the art algorithms and techniques (e.g. provided by commercial suppliers such as GeneArt, GeneCust, GenScript or DNA2.0) that the DNA sequence encoding the leader sequence and/or POI can be optimized to use the codons preferred by the host organism.

In another preferred embodiment, the yeast expression vector or assembly is capable of stably integrating into the yeast genome, for example, by homologous recombination.

A preferred aspect of the invention relates to such a method, wherein the expression vector or assembly comprises a leader sequence effective to cause secretion of the mature POI by the transformed host cell.

The signal sequence or truncated leader sequence of the present invention can be fused to a nucleotide sequence encoding a POI for recombinant expression using conventional cloning techniques known to those skilled in the art. In a preferred embodiment, the nucleotide sequence of the POI is fused to the nucleotide sequence of a secretory leader sequence, whereby the signal sequence or truncated leader sequence targets the protein to the secretory pathway, where the leader sequence is cleaved and the protein is released into the culture medium.

The transformed host cells of the present invention obtained by transforming cells with the expression vectors or components of the present invention can preferably be first cultured under efficient growth conditions to obtain large numbers of cells without the burden of expressing the POI. When preparing a cell line for POI expression, the culture technique is selected to produce the expression product.

This differentiated fermentation strategy can distinguish between a growth phase and a production phase. Growth and/or production can suitably occur in batch mode, fed-batch mode, or continuous mode. Any suitable bioreactor can be used, including batch, fed-batch, continuous, stirred tank reactors, or airlift reactors.

It may be advantageous to provide a pilot scale or industrial scale fermentation process. The industrial scale process may preferably employ a volume of at least 10 L, especially at least 50 L, preferably at least 1 m ³ , preferably at least 10 m ³ , most preferably at least 100 m ³ .

Industrial scale production conditions are preferred, meaning for example fed-batch cultures with reactor volumes of 100 L to 10 m ³ or more, with typical processing times of several days, or continuous processes with fermentation volumes of about 50-1000 L or more and dilution ratios of about 0.02-0.4 ^{h −1} .

Suitable cultivation techniques may include cultivation in a bioreactor, starting with a batch phase, followed by a short exponential fed-batch phase (high specific growth rate), followed by a fed-batch phase at a lower specific growth rate. Another suitable cultivation technique may include a batch culture followed by a continuous culture phase at a lower dilution rate. A preferred embodiment of the present invention includes a batch culture to provide biomass, followed by a fed-batch culture to obtain a high yield of POI.

Preferably, the host cell lines of the present invention are cultured in a bioreactor under growth conditions to achieve a cell density of at least 1 g/L dry cell weight, more preferably at least 10 g/L dry cell weight, and even more preferably at least 20 g/L dry cell weight. Advantageously, pilot-scale or industrial-scale biomass production is provided.

It will be appreciated that the methods disclosed herein may further comprise culturing the recombinant host cell under conditions that allow expression of the POI. A membrane-bound or soluble recombinantly produced POI or host cell metabolite may then be isolated from the cell culture medium and further purified by methods well known to those skilled in the art.

Several different methods are preferred for expressing and secreting POIs from host cells. Proteins are expressed, processed, and secreted by: transforming a eukaryotic organism with an expression vector or assembly of the invention containing DNA encoding the desired protein, preparing a culture of the transformed organism, growing the culture, and recovering the protein from the culture medium, for example, by concentrating and enriching a small portion of the cell culture for the protein, or purifying the protein to obtain a substantially pure preparation. Host cells that have been deleted for one or more major contaminating host cell proteins (e.g., as described in Heiss et al. 2012; Appl Microbiol Biotechnol. doi:10.1007/s00253-012-4260-4) can also be used to facilitate purification of the POI.

As separation and purification methods for obtaining recombinant polypeptide or protein products, methods such as methods utilizing solubility differences, such as salting out and solvent precipitation, methods utilizing molecular weight differences, such as ultrafiltration and gel electrophoresis, methods utilizing charge differences, such as ion exchange chromatography, methods utilizing specific affinity, such as affinity chromatography, methods utilizing hydrophobicity differences, such as reversed-phase high performance liquid chromatography, and methods utilizing isoelectric point differences, such as isoelectric focusing, can be used.

Highly purified products are substantially free of contaminating proteins, preferably having a purity of at least 90%, more preferably at least 95%, or even at least 98%, up to 100%.Purified products can be obtained by purifying cell culture supernatants or from cell debris.

The isolated and purified POI can be identified by conventional methods such as Western blotting or activity assays. The structure of the purified compound can be determined by amino acid analysis, amino terminal analysis, primary structure analysis, and the like. Preferably, the compound can be obtained in large quantities at a high purity, thereby meeting the requirements for use as an active ingredient in a pharmaceutical composition.

Preferred host cell lines of the present invention retain the integration of the regulatory sequences and POI genes of the present invention, and expression levels remain high, for example at least at the μg/L level, even after about 20 generations of culture, preferably after at least 30 generations of culture, more preferably after at least 40 generations, and most preferably after at least 50 generations. Such recombinant host cells are surprisingly stable, which is a great advantage in industrial-scale protein production.

The present invention is further illustrated in detail with reference to the following examples, which are not intended to limit the scope of the present invention in any way.

Examples

Example 1: Construction of a Pichia pastoris Epx1 natural leader sequence (SEQ ID 8) for secretion Pichia pastoris host cell lines for expressing recombinant proteins

1a): Constructing a pichia pastoris Epx1 natural leader sequence (signal sequence and original sequence; SEQ ID 8, EpxL-RT, “leader sequence”) expression vector

The identification of the naturally secreted protein Epx1 in Pichia pastoris and the identification of the putative secretory leader sequence EpxL-RT (composed of the Epx1 signal sequence and the prosequence up to the experimentally determined N-terminus of the mature Epx1 protein) are described in EP 2258855. Since the last amino acid before the N-terminus of mature Epx1 was experimentally confirmed to be Arg-Thr (RT), the putative secretory leader sequence was named EpxL-RT.

To generate an expression vector for secreted POI using the secretory leader sequence of the Pichia pastoris Epx1 protein, the corresponding sequence (SEQ ID 8) was cloned into the pPM2_pGAP expression vector in frame with the PGAP promoter (glyceraldehyde-3-phosphate dehydrogenase promoter). The pPM2_pGAP expression vector is a derivative of the pPuzzle_zeoR_AOXTT vector backbone (described in WO 2008/128701 A2) and consists of the pUC19 bacterial replication origin, the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase promoter, the Saccharomyces cerevisiae CYC1 transcription terminator, and the zeocin antibiotic resistance component.

The Epx1 secretion leader sequence was amplified from Pichia pastoris genomic DNA by PCR using oligonucleotide primers (Table 1).

Table 1: Oligonucleotide primers used for PCR amplification of the leader sequence EpxL-RT of the Pichia pastoris Epx1 protein (restriction sites are underlined)

Subsequently, the PCR product (189 bp) was digested with the restriction enzymes SbfI and NsiI and ligated into the pPM2_pGAP vector, which had been linearized with SbfI and treated with calf intestinal phosphatase (CIP). Due to the overlapping ends generated by NsiI and SbfI, the resulting construct pPM2_pGAPxLRT had a single SbfI site directly before the start codon of the EpxL-RT sequence. Correct integration was verified by digesting the plasmid obtained with SbfI and AscI.

1b) The pPM2_pGAPxLRT vector was used to construct a Pichia pastoris strain that secretes recombinant porcine trypsinogen.

A codon-optimized artificial gene (Geneart, Germany) was synthesized to express recombinant porcine trypsinogen (rpTRP). Using the transport plasmid as a template and the primers shown in Table 2.1, sites for the restriction enzymes Pfl23II and SfiI were added to flank the open reading frame during PCR amplification. The PCR product was digested with Pfl23II and SfiI and cloned into the plasmid pPM2_pGAPxLRT (Example 1a) treated with Pfl23II, SfiI, and CIP. The ligated plasmid was transformed into Escherichia coli (E. coli) TOP10 (Invitrogen) and plated on LB-agar containing bleomycin. Restriction enzyme analysis was performed to confirm the correct properties of the plasmid pPM2_pGAPxLRT_rpTRP.

Table 2.1: Oligonucleotide primers used for PCR amplification of porcine trypsinogen gene

1c) Use pPM2_pGAPxLRT vector to construct a vector that overexpresses recombinant human serum albumin or enhanced green fluorescent protein White Pichia pastoris strain.

The genes encoding human serum albumin (HSA) or enhanced green fluorescent protein (eGFP) were amplified by PCR from vectors described in Stadlmayr et al. (2010, J Biotechnol. 150:519-529) using primers shown in Table 2.2. The PCR products were digested with AccI and SfiI and cloned into the plasmid pPM2_pGAPxLRT treated with AccI and SfiI (Example 1a). The ligated plasmids were transformed into Escherichia coli TOP10 (Invitrogen) and plated on LB-agar containing bleomycin. After sequence confirmation, the vectors pPM2_pGAPxLRT-HSA and pPM2_pGAPxLRT-eGFP were linearized in the promoter region and transformed into Pichia pastoris. For HSA, the construct using the native HSA leader sequence described in Stadlmayr et al. 2010 was used as a reference, while eGFP cloned after the S. cerevisiae MFα leader sequence served as a control.

Table 2.2: Oligonucleotide primers used for PCR amplification of HSA and eGFP fused to EpxL-RT and eGFP fused to the S. cerevisiae MFα leader sequence

Example 2: Cultivation of Pichia pastoris host cell lines for expression of recombinant secretory proteins and product analysis Analysis

Before electrotransformation into Pichia pastoris, all plasmids were linearized at their respective promoter regions or AOX-TT integration regions. Positive transformants were selected on YPD agar plates (comprising yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L) and bleomycin).

2a) Cultivation of transformed Pichia pastoris strains expressing recombinant secreted proteins in small-scale cultures

The pichia pastoris strain single colony from example 1b, 5,7 or 9 is inoculated into 5mLYP- culture medium (10g/L yeast extract, 20g/L peptone) comprising 10g/L glycerol and is cultivated at 28 DEG C overnight. The culture (corresponding final OD ₆₀₀ is 0.1) of packing is transferred to the 10mL expression culture medium (the culture medium composition of each recombinant protein is given below) supplemented with 20g/L glucose and is placed in 100mL conical flask and is cultivated 48h under 170rpm, 28 DEG C of conditions. Alternatively, 2mL of expression culture medium is used to cultivate in 24 deep-well plates. Every 12h repeats to add glucose (10g/L), until harvested cells, by being centrifuged at room temperature for 10min harvested cells and prepared for analysis at 2500xg. pG1-driven expression was achieved by using glucose feed beads (Kuhner, CH) to achieve glucose-limited growth conditions. These feed beads slowly release glucose over time according to the equation (glucose) = 1.63*t ^0.74 [mg/plate], without glucose supplementation. Two feeder beads were used for 10 mL of the main culture. Biomass was determined by measuring the cell weight after centrifugation of 1 mL of cell suspension, and recombinant secreted protein in the supernatant was determined as described in Examples 2b-2e below.

The expression medium is as follows:

For porcine trypsinogen: per liter: 10 g yeast extract, 10 g pea peptone, 10.2 g (NH4) ₂ PO ₄ , 1.24 g KCl, 0.1 g CaCl ₂ , adjust pH to 5.0 with HCl

For HSA: per _liter : 22 g citric acid, 3.15 _g ( _NH4 ) _2HPO4 , 0.027 _g CaCl2*2H2O, 0.9 g KCl, 0.5 g _MgSO4 * _7H2O , 2 ml 500x biotin, and 1.47 mL trace salt stock solution _[ per liter: ₆ g CuSO4* _5H2O , 0.08 g _NaI , 3 g MnSO4* _H2O , 0.2 _{g Na2MoO4} *2H2O, _0.02 g _H3BO3 , 0.5 _g CoCl2, 20 g ZnCl2, ₅ g FeSO4* _7H2O , and 5 _{mL H2SO4} ]; set pH to 6 with 5 _M KOH; filter sterilize.

For eGFP and for antibody fragments (e.g., Fab): Per liter: 10 g yeast extract, 10 g peptone, 100 mM potassium phosphate buffer, pH 6.0, 13.4 g yeast nitrogenous base and ammonium sulfate, 0.4 mg biotin

2b) Trypsin quantification

Pichia pastoris culture supernatant was desalted using a small, prepacked molecular sieve chromatography column (Disposable PD-10 Desalting Column 17-0851-01; GE Healthcare). 2.5 mL of supernatant was applied to the column and eluted with 3.5 mL of 1 mM HCl. Following elution, 70 μL of ₂ M CaCl₂ solution was added.

To convert inactive trypsinogen into active trypsin, 300 μL of desalted supernatant (+CaCl ₂ ) was mixed with 690 μL of activation buffer (50 mM TRIS/HCl pH 8.6; 40 mM CaCl ₂ and 0.15 g/L enterokinase, Sigma; E0632) and incubated at 37° C. for 2 h.

165 μL of the activation mixture was mixed with 1000 μL of TAME solution containing 446 mg/L Na-p-tosyl-L-arginine-methyl-ester-hydrochloride (TAME; Sigma; T4626) dissolved in dilution buffer (50 mM TRIS/HCl pH 8.1; 40 mM CaCl ₂ ) and the absorption kinetics at 247 nm were measured in a spectrophotometer at 30° C. over a period of 5 minutes. If necessary, the activated trypsin solution was diluted with dilution buffer to reach the linear range of the method (ΔA247/min <0.3). A 1 g/L trypsin concentrate corresponds to a ΔA247/min = 0.101.

2c) Quantification of HSA by ELISA

To quantify HSA in Pichia pastoris supernatants, a human serum albumin ELISA kit (Cat. No. E80-129, Bethyl Laboratories, TX, USA) was used. HSA standards were used at a starting concentration of 400 ng/mL. Supernatant samples were diluted accordingly.

2d) SDS-PAGE & Western blot analysis

Use Bis-Tris system for protein gel analysis, it uses 12% Bis-Tri or 4-12% Bis-Tris gel and MOPS running buffer (all from Invitrogen).After electrophoresis, protein is visualized by silver staining, or is transferred to nitrocellulose filter and is used for western blot analysis.Therefore, use the XCell II ^TM blotting module (Invitrogen) that is used for wet formula (trough) transfer according to the manufacturer's specification sheets that protein is electrotransferred to nitrocellulose filter. After blocking, Western blots were probed with the following antibodies: for HSA: anti-human serum albumin-horseradish peroxidase (HRP) conjugate, Bethyl A80-129P (1:50,000); for IgG light chain: anti-human kappa light chain (bound or free)-alkaline phosphatase (AP) conjugate antibody, Sigma A3813 (1:5,000); for IgG heavy chain: anti-human IgG (γ chain specific) antibody raised in goat, Sigma I3382 (1:5,000) and anti-goat AP conjugate (1:20,000).

AP conjugates were detected using a colorimetric AP detection kit (BioRad) based on the NBT/BCIP system, and HRP conjugates were detected using the chemiluminescent Super Signal West chemiluminescent substrate (Thermo Scientific).

2e) Quantification of Fab by ELISA

Intact Fab was quantified by ELISA using an anti-human IgG antibody (Abcam ab7497) as the coating antibody (1:1,000) and a goat anti-human kappa light chain (bound or free) alkaline phosphatase-conjugated antibody (Sigma A3813) as the detection antibody (1:1,000). A human Fab/κ, IgG fragment (Bethyl P80-115) was used as a standard at a starting concentration of 50 ng/mL. Supernatant samples were diluted accordingly. Detection was performed using pNPP substrate (Sigma S0942 ₎ . Coating, dilution, and wash buffers were based on PBS (2 mM _KH2PO4 , 10 mM _Na2HPO4.2H2O , 2.7 _mM g KCl, 8 mM NaCl, pH 7.4) and included BSA (1 _% (w/v)) and/or Tween 20 (0.1% (v/v)), as appropriate.

Example 3: Using the Pichia pastoris Epx1 leader sequence for secretion (EpxL-RT, SEQ ID 8) Pichia pastoris host cell lines expressing recombinant proteins

3a) Analysis of Pichia pastoris overexpressing recombinant porcine trypsinogen using EpxL-RT for secretion

Pichia pastoris expressing pTRP using the EpxL-RT sequence for secretion was cultured as described in Example 2a and analyzed using a trypsin activity assay (described in Example 2b) and SDS-PAGE (described in Example 2d). Pichia pastoris expressing pTRP using MFα for secretion was used as a reference.

Unexpectedly, expression of pTRP using the EpxL-RT secretory leader sequence resulted in a protein tail of approximately 30 kDa (Figure 2.1, left), whereas MFα resulted in secreted pTRP of the correct size (25 kDa; Figure 2.1, right). The partial pTRP secreted when using EpxL-RT also resulted in pTRP of the correct size, albeit to a lesser extent. The amount of pTRP secreted when using EpxL-RT was less than 50% of the amount secreted when using MFα (Table 3, determined by the trypsin activity assay described in Example 2b). The tailing band is likely an EpxL-RT-pTRP fusion protein, which appears to be caused by improper processing (cleavage) of the EpxL-RT sequence.

Table 3: Relative secretion levels of pTRP using αMF as a standard

leader sequence The relative mean secretion of pTRP ± SEM EpxL-RT 0.48±0.02 MFα 1.00±0.05

3b) Analysis of Pichia pastoris overexpressing recombinant eGFP using EpxL-RT for secretion

Using the secreted EpxL-RT sequence, Pichia pastoris expressing eGFP was cultured as described in Example 2a and analyzed using SDS-PAGE and Western blotting (Example 2d). Clearly, compared to MFα (Figure 2.2, right panel), native EpxL-RT was unable to fully secrete eGFP (Figure 2.2, left panel). In the cells, eGFP was observed to remain partially bound to the EpxL-RT sequence, thus ruling out defects in protein expression (not shown).

3c) Separation of Pichia pastoris overexpressing recombinant human serum albumin using EpxL-RT for secretion Analysis.

Pichia pastoris expressing HSA using the secreted EpxL-RT sequence was cultured as described in Example 2a and analyzed using HSA ELISA (Example 2c) and Western blot (Example 2d). Pichia pastoris expressing HSA using the native HSA leader sequence for secretion was used as a reference (Kobayashi. 2006. Biologicals. 34(1):55-9.).

After 48 h of culture in shake flasks in synthetic-screening medium, the secretion behavior of 12 individual clones of each construct was analyzed. The supernatant was qualitatively examined by reducing SDS-PAGE and subsequent Western blotting using an anti-HSA antibody for detecting HSA. A clear double-band pattern was seen for HSA secreted by EpxL-RT (Figure 2.3, left). Compared with HSA secreted from its native leader sequence (Figure 2.3, right) and purified HSA (not shown), the lower band is correctly processed HSA. The upper band was unexpected, but since its molecular weight is only slightly larger than the lower band, it may represent an EpxL-RT-HSA fusion protein caused by incorrect processing of EpxL-RT. Based on the band density (analyzed by ImageJ), 40-60% of the total amount of secreted HSA exists in an incorrect, higher molecular weight form.

Summary: When using the EpxL-RT sequence for secretion, a smearing or double band pattern can be seen for the secreted recombinant protein, indicating that the long leader sequence EpxL-RT is not processed correctly or not processed at all. This feature makes the EpxL-RT corresponding to the full-length Epx1 leader sequence of EP2258855A1 unsuitable and cannot be used for recombinant protein secretion.

Example 4: N-terminal identification of higher molecular weight bands from HSA expressing cultures

The N-terminus of HSA expressed when using EpxL-RT as described in Example 3c) above was then further subjected to N-terminal sequencing.

Therefore, 500 μL of the corresponding supernatant was loaded onto a centrifugal filter for protein concentration (Amicon Ultra-0.5 mL 10 kDa centrifugal filter, Millipore, UFC5010), centrifuged for 5 min, and 15 μL of sample was recovered by reverse spinning.

The samples were then separated by 4-12% Bis-Tris NuPAGE gel and subjected to Western blotting using a PVDF (polyvinylidene fluoride) membrane at 25 V for 2 hours using borate buffer (per liter: 3.09 g borate [50 mM], 100 mL MeOH, pH set to 9 with 1 M NaOH). Prior to blotting, the gel was incubated in borate buffer for 10 minutes, and the membrane was immersed in methanol for 30 seconds and then immersed in borate buffer for 3 minutes.

After blotting, the membrane was stained with Coomassie Brilliant Blue (0.1% [w/v] R250, methanol [40% v/v], acetic acid [10% v/v]) for 3 minutes, followed by destaining (methanol [40% v/v], acetic acid [10% v/v]). The membrane was rinsed with _ddH₂O , and the upper and lower bands were excised (Figure 3) and submitted for N-terminal EDMAN sequencing. For the lower band, D was identified as the N-terminal amino acid, ultimately containing the first amino acid of HSA (HSA N-terminus, SEQ ID 30: DAHKSEV), while for the upper band, AYYT (SEQ ID 31) was identified as the N-terminus of the secreted protein. These amino acids are part of the EpxL-RT sequence (SEQ ID 8), leaving an unnecessary 21 amino acid overhang on HSA. The EpxL-RT sequence preceding AYYT (SEQ ID 31) is KR, which is likely part of a binary Lys-Arg peptidase cleavage motif. Processing of the binary Lys-Arg motif by proteases such as Kex2 depends not only on the motif itself, but also on the three-dimensional structure and surrounding amino acid environment (Bader et al. 2008, BMC Microbiol. 8:116). BLAST analysis and sequence alignment of EpxL-RT with Epx1 homologs from other yeasts (e.g., Saccharomyces cerevisiae and Candida albicans) revealed that the KR motif in the leader sequence is non-conserved. Therefore, processing of the KR motif by proteases such as Kex2 cannot be considered a priori based on sequence analysis.

Example 5: Use of the Pichia pastoris EpxL-KR sequence for secretion (truncated leader sequence, SEQ ID 10, wherein X at position 3 is F and X at position 16 is A (SEQ ID 12)) Construction of a Pichia pastoris host cell line for expression recombinant protein

To test whether the KR sequence in EpxL-RT is a protease cleavage site, the EpxL-KR sequence for secretion was used to construct a vector for expressing recombinant protein.

5a) Construction of a Pichia pastoris strain overexpressing HSA using the EpxL-KR sequence for secretion.

HSA was amplified using the primers in Table 4.1 and ligated into the vector pPM2_pGAPxLRT digested with Bgl I and Sbfi I. After sequence confirmation, the vector pPM2_pGAPxLKR-HSA was linearized in the promoter region and transformed into Pichia pastoris.

Table 4.1: Oligonucleotide primers for PCR amplification of HSA fused to EpxL-KR (restriction sites are underlined, EpxL sequence is italicized)

5b) Construction of Pasteurella overexpressing porcine trypsinogen or eGFP using the EpxL-KR sequence for secretion Trichoderma yeast strains.

pTRP was amplified using the primers in Table 4.2 and ligated into the pPM2_pGAPxLRT vector digested with PvuII and SbfiI. The expression vector pPM2_pGAPxLKR-eGFP was generated using the same method. After sequence confirmation, the pPM2_pGAPxLKR-pTRP and pPM2_pGAPxLKR-eGFP vectors were linearized at the promoter region and transformed into Pichia pastoris.

Table 4.2: Oligonucleotide primers for PCR amplification of pTRP or eGFP fused to EpxL-KR (restriction sites are underlined, EpxL sequence is italicized)

5c) Construction of Pichia pastoris overexpressing antibody light or heavy chains using the EpxL-KR sequence for secretion mother plant.

The light chain (LC) and heavy chain (HC) of the antibody HyHEL were amplified using the primers in Table 4.3 and ligated to the vector pPM2_pGAPxLRT digested with BglII and SbfiI. After sequence confirmation, the vectors pPM2_pGAPxLKR-LC and pPM2_pGAPxLKR-HC were linearized in the promoter region and transformed into Pichia pastoris.

Table 4.3: Oligonucleotide primers for PCR amplification of HyHEL LC or HC fused to EpxL-KR (restriction sites are underlined, EpxL sequence is italicized)

Example 6: Use of EpxL-KR for secretion (truncated leader sequence, SEQ ID 10, wherein X at position 3 is F, The recombinant protein was expressed in a Pichia pastoris host cell line.

6a) Separation of Pichia pastoris overexpressing recombinant porcine trypsinogen using EpxL-KR for secretion Analysis

Pichia pastoris expressing pTRP (Example 5b) using the EpxL-KR sequence for secretion was cultured as described in Example 2a and analyzed as described in Example 3a.

Surprisingly, we observed that the use of the shortened Epx secretion leader sequence, EpxL-KR (Figure 4.1, middle), indeed enhanced processing. In contrast to the protein tailing observed when using EpxL-RT for secretion (Figure 4.1, left panel), pTRP secreted using EpxL-KR produced a band of the correct size (Figure 4.1, middle panel). The correct N-terminus of pTRP secreted using EpxL-KR was verified by mass spectrometry analysis (LC-MS).

Therefore, approximately 10 μg of each sample was separated by SDS-PAGE, the desired protein band was cut out and digested in the gel. The protein was carbamidomethylated in the gel. The protein was digested with sequencing grade trypsin (Roche), or with Glu-C (Sigma-Aldrich), LysC (Roche) and chymotrypsin (Chymotrypsin) (Roche), or analyzed directly without digestion. All proteolytic reactions were carried out at 37°C overnight. After this, the sample was directly injected into the LC-MS system (LC: Dionex Ultimate 3000LC, MS: Bruker, Amazon ETD, equipped with an online nano source). Peptides were separated on a C-18 column (Dr. Maisch GmbH, C-18 HPLC column ReproSil-Pur 200*0.1 mm, 3 μm packing, 200 Å pore size, flow rate: 0.4 μL/min) using a linear gradient from 95% solvent A and 5% solvent B (solvent A: 0.1% FA in water, 0.1% FA in ACCN) to 32% B over 40 min, followed by a 15-min linear gradient from 32% B to 75% B to facilitate elution of large peptides. Analysis was performed using MS with data-dependent acquisition. Data were processed using standard Bruker software (data analysis) and the freeware program X!-tandem in conjunction with GPM.

In contrast to the correct N-terminus of pTRP secreted with EpxL-KR, the amino acid EAEA was confirmed to remain at the N-terminus of secreted pTRP when MFα was used for secretion. Furthermore, the amount of pTRP secreted using EpxL-KR was more than 20% higher than that of the commonly used MFα secretion leader sequence (Table 5).

Table 5: Relative secretion levels of pTRP using MFα as standard

leader sequence The relative mean secretion of pTRP ± SEM EpxL-KR 1.21±0.02 MFα 1.00±0.09

6b) Separation of Pichia pastoris overexpressing recombinant human serum albumin using EpxL-KR for secretion Analysis

Pichia pastoris expressing HSA using the EpxL-KR sequence for secretion (Example 5a) was cultured as described in Example 2a and analyzed using Western blotting (Example 2d).

In contrast to the double band pattern observed when using EpxL-RT for secretion (Figure 2.3, left panel), HSA secreted using EpxL-KR produced a single band of the correct size (not shown). Thus, EpxL-KR is able to secrete correctly processed HSA.

6c) Analysis of Pichia pastoris overexpressing eGFP using EpxL-KR for secretion

Pichia pastoris expressing eGFP (Example 5b) using the EpxL-KR sequence for secretion was cultured as described in Example 2a and analyzed as described in Example 3b.

Only the EpxL-KR leader sequence resulted in eGFP of the correct size (Figure 4.2, left), while the remaining MFα leader sequence resulted in eGFP of a higher molecular weight (Figure 4.2, right). LC-MS analysis (described in Example 6a) confirmed the correct N-terminus of the eGFP secreted by EpxL-KR, demonstrating that the content of molecules with the correct N-terminus in the secreted product was at least 95%, preferably at least 98%, even more preferably at least 99%, or approximately 100% (w/w). In contrast, the use of MFα resulted in the amino acids EAEA being retained as an additional amino acid sequence at the N-terminus due to incorrect processing of the leader peptide by the Ste13 aminopeptidase. Based on the band sizes on SDS-PAGE, the majority of the eGFP secreted by MFα possessed the incorrect amino acid overhang EAEA at the N-terminus, whereas no incorrectly sized bands were observed for the eGFP secreted by EpxL-KR. For pTRP (Example 6a), the secretion level of eGFP was greater than 20% higher with EpxL-KR compared to that with MFα (Table 6).

Table 6: Relative secretion levels of eGFP using MFα as a standard (quantified by band density using ImageJ software)

leader sequence Relative mean secretion of eGFP ± SEM EpxL-KR 1.23±0.05 MFα 1.00±0.08

6d) Using EpxL-KR for secretion to overexpress antibody light chain or antibody heavy chain Pichia pastoris Analysis

Pichia pastoris expressing HyHEL LC or HyHEL HC using the EpxL-KR sequence for secretion (Example 5c) was cultured as described in Example 2a and analyzed as described in Example 2b.

Both antibody chains could be secreted using the EpxL-KR sequence (Figure 4.3), confirming that EpxL-KR is a valuable secretion leader sequence for antibody production in Pichia pastoris. The correct N-terminus of HyHEL LC secreted by EpxL-KR was verified by LC-MS analysis (as described in Example 6a).

Example 7: Construction of Pichia pastoris host cells using the EpxL-AA sequence (SEQ ID 21) for secretion Used to express recombinant proteins

WO2010/135678 and US20110021378 describe the amino acid sequence MKLSTNLILAIAAASAVVSAA (SEQ ID 21), i.e., amino acids 1-21 of SEQ ID 8, which correspond to the first 21 amino acids of the full-length Epx1 leader sequence. However, WO2010/135678 and US20110021378 do not provide experimental data demonstrating that this sequence is actually suitable for secretion of recombinant proteins. To test whether this fragment (hereinafter referred to as EpxL-AA) is sufficient to secrete correctly processed recombinant proteins, we constructed a vector for expressing recombinant proteins using the secreted EpxL-AA sequence.

Antibody light chain LC, pTRP, and eGFP were amplified using the primers in Table 7 and ligated to the vector pPM2_pGAPxLRT digested with BglI and SfiI. After sequence confirmation, the vectors pPM2_pGAPxLAA-LC, pPM2_pGAPxLAA-pTRP, and pPM2_pGAPxLAA-eGFP were linearized in the promoter region and transformed into Pichia pastoris, respectively.

Table 7: Oligonucleotide primers for PCR amplification of pTRP, eGFP and HyHEL LC fused to EpxL-AA (restriction sites are underlined, EpxL sequence is italicized)

Example 8: Expression from a Pichia pastoris host cell line using EpxL-AA (SEQ ID 21) for secretion recombinant protein

8a) Analysis of Pichia pastoris overexpressing eGFP using EpxL-AA for secretion

Pichia pastoris expressing eGFP (Example 7) was cultured as described in Example 2a using EpxL-AA for secretion and analyzed as described in Example 3b.

On SDS-PAGE, the band with EpxL-AA was slightly larger than that of EpxL-KR ( FIG. 5.1 ), indicating that at least one alanine residue was left at the N-terminus of eGFP, thus representing a non-native N-terminus of the recombinant secreted protein.

8b) Separation of Pichia pastoris overexpressing recombinant porcine trypsinogen using EpxL-AA for secretion Analysis

Pichia pastoris expressing pTRP (Example 7) using the EpxL-AA sequence for secretion was cultured as described in Example 2a and analyzed as described in Example 3a.

pTRP was secreted using the EpxL-AA sequence, however, the secretion level was lower than that of the EpxL-KR sequence (Table 8). On SDS-PAGE, the band with EpxL-AA was slightly larger than that with EpxL-KR (Figure 5.1), indicating that at least one alanine residue was left at the N-terminus of pTRP.

Indeed, N-terminal sequencing of the EpxL-AA secreted pTRP revealed that the N-terminus of the recombinant secreted protein contained an additional amino acid, Ala, which was retained from the EpxL-AA sequence, thus representing a non-native N-terminus of the recombinant secreted protein.

Table 8: Relative secretion levels using EpxL-KR as standard

leader sequence The relative mean secretion of pTRP ± SEM EpxL-KR 1.00±0.12 EPxL-AA 0.79±0.04

8c) Analysis of LC-overexpressing Pichia pastoris using EpxL-AA for secretion

Pichia pastoris expressing HyHEL LC (Example 7) using the secreted EpxL-AA sequence was cultured as described in Example 2a and analyzed as described in Example 2d. For LC, secretion levels were lower when EpxL-AA was used than when EpxL-KR or MFα were used (Figure 5.2). For pTRP, N-terminal sequencing of the LC secreted by EpxL-AA revealed that the N-terminus of the recombinant secreted protein contained an additional amino acid, Ala, which was retained from the EpxL-AA sequence.

This is again an unwanted residue from the signal sequence, making EpxL-AA unsuitable for production of secreted recombinant protein.

Example 9: Using the EpxL-A sequence for secretion (signal peptide sequence, SEQ ID 1, wherein X at position 3 is F, Where X is A (SEQ ID 3)) Construction of a Pichia pastoris host cell line for expression of recombinant protein

Therefore, we constructed a Pichia pastoris strain that secretes recombinant proteins via a signal sequence consisting of only the first 20 amino acids of the native Epx1 leader sequence, termed EpxL-A. Ala represents the last amino acid of the predicted signal peptidase cleavage site (C-terminus), while the recombinant protein begins immediately on the N-terminal side of the SP cleavage site. To verify that the nature of this N-terminal amino acid does not affect SP cleavage, we tested the secretion of three different recombinant proteins: eGFP (starting with the hydrophobic amino acid Val) and the antibodies LC and HC (starting with the negatively charged amino acid Asp).

Antibody light chain LC, Fab-HC and eGFP were amplified using the primers in Table 9 and ligated to the vector pPM2_pGAPxLRT digested with BglI and Sfil. After sequence confirmation, the vector was linearized in the promoter region and transformed into Pichia pastoris.

For LC and Fab-HC, the pGAP promoter was also exchanged for the inducible pG1 promoter (SEQ ID 9) using ApaI and SbfI, and the expression components of both chains were then combined into a single vector using compatible restriction enzymes MreI and AgeI. After sequence confirmation, the vector was linearized at the AOX-TT region and transformed into Pichia pastoris.

Table 9: Oligonucleotide primers for PCR amplification of eGFP and HyHEL LC and Fab-HC fused to EpxL-A (restriction sites are underlined, EpxL sequence is italicized)

Example 10: Use of EpxL-A for secretion (signal peptide sequence, SEQ ID 1, wherein X at position 3 is F, 16 where X is A (SEQ ID 3)) was expressed as a recombinant protein in a Pichia pastoris host cell line.

10a) Analysis of Pichia pastoris overexpressing eGFP using EpxL-A for secretion

Pichia pastoris expressing eGFP (Example 9) using the EpxL-A sequence for secretion was cultured as described in Example 2a and analyzed as described in Example 3b.

As can be seen from FIG5.1 , the secretion level of eGFP using EpxL-A was comparable to that using EpxL-KR and higher than that using EpxL-AA.

In contrast to the hydrophobic signal and leader sequence described by Kottmaier et al. (2011 Appl Microbiol Biotechnol. 91: 133-141) and the pre-pro leader sequence of MFα, the EpxL-A sequence and the EpxL-KR sequence did not result in vacuolar targeting of eGFP (confirmed by confocal laser scanning microscopy - data not shown). Both cell lines showed intracellular distribution of eGFP, representing a protein present in the secretory pathway (primarily endoplasmic reticulum staining); therefore, the EpxL-A signal peptide and the EpxL-KR truncated leader sequence appear to be very suitable for secretion of recombinant proteins, as they exhibit a "secretory phenotype."

10b) Analysis of LC-overexpressing Pichia pastoris using EpxL-A for secretion

Pichia pastoris expressing HyHEL LC (Example 9) using the EpxL-A sequence for secretion was cultured as described in Example 2a and analyzed as described in Example 6d.

The secretion of the antibody light chain using EpxL-A resulted in a protein of the correct size (Figure 5.2), and thus the secretion efficiency was similar to that using EpxL-KR and more than 8 times higher than that using EpxL-AA (Table 10). The secretion levels were compared by quantifying the band density on the gel shown in Figure 5.2 using ImageJ software.

Table 10: Relative secretion levels of HyHEL LC using EpxL-AA as standard.

leader sequence Relative mean secretion of LC ± SEM EpxL-KR 8.82±0.53 EPxL-AA 1.00±0.55 EPxL-A 9.53±0.87

The N-terminus of the LC secreted from EpxL-A was verified by LC-MS to be the correct N-terminus DIVLTQSP (Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro, SEQ ID 54).

Therefore, in contrast to the longer sequence EpxL-AA, the signal peptide EpxL-A of Epx1 is sufficiently suitable for secretion of the recombinant protein with the correct N-terminus.

In addition to testing expression under the control of the constitutive pGAP promoter, LC secretion with EpxL-A under the control of the inducible pG1 promoter (SEQ ID 9) was also tested. Cell line construction was as described in Example 9. Glucose-limited conditions were generated in screening cultures using glucose feeder beads as described in Example 2a. Under these inducing conditions, light chain secretion was observed.

10c) Analysis of Pichia pastoris overexpressing antibody Fab fragments using EpxL-A for secretion

Pichia pastoris (cell strain described in Example 9) expressing the Fab fragment of the HyHEL antibody, consisting of light chain ( _vL and _cL ) and heavy chain fragment ( _vH and _cHI ), using the EpxL-A sequence for secretion of both chains, was cultured as described in Example 2a and analyzed as described in Examples 2d and 2e.

Surprisingly, when the EpxL-A sequence was used for the LC and HC secretion of HyHEL Fab, the level of intact secreted Fab was up to 10-fold higher than when the MFα prepro leader sequence was used (determined by ELISA as described in Example 2e). When EpxL-A was used, the Fab yield per unit of biomass was 0.15-0.35 mg Fab/OD, while when MFα was used for secretion, it was 0.03-0.13 mg Fab/OD. Moreover, the supernatant of Pichia pastoris secreting HyHEL Fab under pG1 control with EpxL-A did not contain high levels of free LC or high molecular weight aggregates of the recombinant protein (Figure 5.3).

Example 11: Use of EpxL-A for secretion (signal peptide sequence, SEQ ID 1, wherein X at position 3 is F, 16 where X is A (SEQ ID 3)) was expressed as a recombinant protein in a Pichia pastoris host cell line.

Eight proteins secreted from Pichia pastoris using EpxL-A (SEQ ID 3) were tested: human growth hormone (HGH), somatotropin, interferon α2a (IFN-α2a), two different histidine-tagged scFvs (scFvs1 and scFvs2), and three different Fabs (Fab1, Fab2, and Fab3).

The gene was codon-optimized and synthesized for Pichia pastoris by GeneArt (Germany). The resulting vector was digested with SbfI and SfiI, and the gene was ligated into the vector pPM2aZ30_pG1. In the case of Fabs, the expression assemblies of both chains were combined into a single vector using the compatible restriction enzymes MreI and AgeI. After sequence confirmation, the vector was linearized at the AOX terminator region and transformed into Pichia pastoris.

A Pichia pastoris strain expressing recombinant proteins human growth hormone, somatotropin, and interferon α2a (IFN-α2a), two histidine-tagged scFvs (scFvs1 and scFvs2), and three Fabs (Fab1, Fab2, and Fab3) (Fabs consisting of light chain ( _vL and _cL ) and heavy chain fragments ( _vH and _cH1 )) using the secreted EpxL-A sequence (SEQ ID 3) was cultured in a medium containing 10 g yeast extract, 10 g peptone, 100 mM potassium phosphate buffer, pH 6.0, 13.4 g yeast nitrogenous base and ammonium sulfate, and 0.4 mg biotin per liter. Small-scale screening was performed in 24-well plates using two feeder beads (Kuhner, 6 mm diameter).

The supernatant was analyzed as described in Example 2d. POI was detected in Western blots using specific antibodies: For somatotropin and human growth hormone: GH1 polyclonal antibody; Proteintech 17867-1AP (1:5,000) and anti-rabbit IgG (whole molecule)-alkaline phosphatase antibody, Sigma A3687 (1:12,000). For interferon α2a: interferon, α2a antibody; Antibodies-Online ABIN573795 (1:1,500) and anti-rabbit IgG (whole molecule)-alkaline phosphatase antibody, Sigma A3687 (1:12,000). For histidine-tagged scFvs: Penta-His HRP conjugate; QIAGEN 10149928 (1:1,500).

When the EpxL-A sequence (SEQ ID 3) was used ( FIG. 6 ), all tested proteins were successfully secreted into the culture medium.

Claims (21)

1. An isolated nucleic acid encoding a leader sequence, said leader sequence being a leader peptide of the amino acid sequence of SEQ ID NO:12, wherein the nucleic acid consists of the nucleotide sequence of SEQ ID NO:19 or a codon-optimized variant of SEQ ID NO:19. 2. An isolated nucleic acid encoding a leader sequence, said leader sequence being a signal peptide of the amino acid sequence of SEQ ID NO:3, wherein the nucleic acid consists of the nucleotide sequence of SEQ ID NO:16 or a codon-optimized variant of SEQ ID NO:16. 3. The nucleic acid according to any one of claims 1-2, wherein the nucleic acid is DNA. 4. The isolated leader sequence encoded by the nucleic acid according to any one of claims 1-2. 5. The leader sequence according to claim 4, wherein the leader sequence is a peptide, the peptide consisting of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:12. 6. An expression component comprising a nucleic acid encoding a leader sequence, said nucleic acid encoding the leader sequence being fused with a nucleic acid sequence encoding a POI, characterized in that the leader sequence is selected from the group consisting of: (a) The leader peptide of the amino acid sequence of SEQ ID NO:12, wherein the nucleic acid encoding the leader sequence is a nucleotide sequence encoding the leader peptide, said nucleotide sequence being composed of the nucleotide sequence of SEQ ID NO:19 or a codon-optimized variant of SEQ ID NO:19. (b) The signal peptide of the amino acid sequence of SEQ ID NO:3, wherein the nucleic acid encoding the leader sequence is a nucleotide sequence encoding the leader peptide, the nucleotide sequence being composed of the nucleotide sequence of SEQ ID NO:16 or a codon-optimized variant of SEQ ID NO:16. 7. The expression component of claim 6, wherein the POI comprises an amino acid sequence having a natural N-terminal amino acid sequence. 8. The expression component according to claim 6 or 7, wherein the POI is selected from the group consisting of: therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine and vaccine-like proteins or particles, process enzymes, growth factors, hormones and cytokines, or wherein the POI mediates the production of host cell metabolites. 9. The expression component of claim 8, wherein the POI is selected from the group consisting of growth factors, hormones, cytokines, antibodies, or antibody fragments. 10. The expression component of claim 9, wherein the POI is selected from the group consisting of: full-length antibodies, scFv, microantibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, Fab and Fc fusion proteins. 11. The expression component according to any one of claims 6-10, further comprising a promoter operatively linked to a nucleic acid encoding a leader sequence. 12. A recombinant yeast host cell or vector comprising the expression component according to any one of claims 6-11. 13. The host cell according to claim 12, wherein the yeast is selected from the group consisting of the genera: Pichia pastoris, Candida, Globosa, Arxula, Hansenula, Ogatea, Yersinia, Kluyveromyces, Yeast, or Komagataella. 14. A method for producing POI in yeast host cells, comprising: - Provide a host cell according to claim 12 or 13, -Culturing the host cells to express the POI, and - Purify POI to obtain purified POI products. 15. The method of claim 14, wherein the POI comprises an amino acid sequence having a natural N-terminal amino acid sequence. 16. The method of claim 14 or 15, wherein the POI comprises an amino acid sequence that does not include an additional alanine as an N-terminal amino acid residue. 17. The method according to any one of claims 14-15, wherein the POI is selected from the group consisting of: therapeutic proteins, including antibodies or fragments thereof, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine and vaccine-like proteins or particles, process enzymes, growth factors, hormones and cytokines, or wherein the POI mediates the production of host cell metabolites. 18. The method of claim 17, wherein the POI is selected from the group consisting of growth factors, hormones, cytokines, antibodies, or antibody fragments. 19. The method of claim 17, wherein the POI is selected from the group consisting of full-length antibodies, scFv, microantibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, Fab and Fc fusion proteins. 20. Use of the nucleic acid according to any one of claims 1-3 or the leader sequence according to any one of claims 4-5 for secreting POI from host cells and/or increasing the secretion of POI from host cells. 21. The use according to claim 20, wherein at least 60% of the secreted POI comprises a natural N-terminal amino acid sequence.