HK1065818B - Supersecretable peptides, processes for their production, and parallel improvement of the secreted form of one or more other polypeptides - Google Patents

Description

Hypersecretoble peptides, methods of making them, and parallel enhancement of the secretory form of one or more other polypeptides

In view of economic viability, the process for producing a pharmaceutically relevant protein must result in a biologically active product having as high a purity as possible. Yeast is widely used herein for the expression of these related proteins. Proteins such as insulin, GM-CSF () And hirudin () Based on the synthesis of specific proteins or their precursors in yeastExamples were developed because of the success of the engineering approach. In general, yeasts are capable of direct, significant synthesis of hirudin with high yields, which are on the gram scale when Hansenula polymorpha (Hansenula polymorpha) (Weydemann et al, appl. Microbiol Biotechnol.44: 377-385, 1995) or Pichia pastoris (Pichia pastoris) (Rosenfeld et al, Protein Expr. purif: 4, 476-82, 1996) is used.

EP-A0324712 describes hirudin derivatives () The N-terminal amino acid is leucine and is constitutively expressed in Saccharomyces cerevisiae strain Y79. EP-A0347781 describes a mini-proinsulin (mini-proinsulin) and its expression in, for example, baker's yeast.And insulin are produced by two separate expressions.

Surprisingly, we have found that hirudin derivatives and miniproinsulin derivatives can be obtained from a common precursor protein by: the precursor protein is fused via a basic dipeptide, preferably Lys-Arg, to a signal or leader sequence recognized by yeast as a secretion signal, and likewise a cleavage site recognized by an endoprotease of yeast is introduced between the N-terminal hirudin derivative and the proinsulin derivative. Here too, basic dipeptides, such as Lys-Arg, are preferred. After expression, the hirudin derivative extended with Lys-Arg and the mini-proinsulin derivative starting with the first amino acid of the insulin B chain are found in the supernatant. Surprisingly, the production of mini-proinsulin obtained by this method is significantly improved compared to the direct signal-mini-proinsulin expression, while the production of hirudin derivatives remains essentially unchanged. Surprisingly, this way hirudin acts as an enhancer peptide for the production of miniproinsulin.

Peptides that can act as enhancing proteins are generally relatively small and can be secreted in a short period of time, abundantly and naturally, from, for example, glandular tissue. Peptides of this type, including for example snake venom or eglin C or TAP (tick anticoagulant peptide) can be distinguished by excellent export compatibility. The invention also relates to such proteins.

A further advantage is that the hirudin derivatives can have the same or better pharmaceutical properties than hirudins already used in medicaments. For this case, it is possible to produce two or even more drugs from one and the same fermentation. Thus, the required fermentation capacity is reduced. This directly contributes to the production costs.

However, it is optional whether to produce multiple products. For example, the need for Refludan is smaller than for insulin, which can lead to methods of discarding one of the pharmaceutically interesting substances.

In order to increase the yield, it is possible, as proposed in patent application EP-A0200655, to place a short peptide sequence as a linker for the signal or leader sequence before the N-terminus of the hirudin derivative via Lys-Arg. It will be apparent to those skilled in the art that the choice of signal or leader sequence also directly affects the yield of the protein of interest. The selection of this sequence is the subject of further optimization. The sequence located at the 3' end of the expression cassette also directly affects the yield by affecting the stability of the mRNA. Here, it is also obvious to the person skilled in the art that the above-described sequences can be optimized for each protein of interest to be expressed. This is also true for the selection of a suitable promoter, which may be inducible or constitutively active. The choice of carrier system and host system is also important for throughput. Thus, in addition to the baker's yeast already exemplified, the yeasts Pichia pastoris (Pichia pastoris), Hansenula polymorpha (Hansenula polymorpha) or Kluyveromyces lactis (K.lactis) and also vectors or expression cassettes which are in each case optimized for different physiological conditions can also be used.

Another advantage of the method allowing secretion into the culture medium is that the protein-chemical work-up of the protein of interest is relatively simple. Surprisingly, we have found that miniproinsulin can be concentrated in the presence of hirudin by membrane filtration, the exclusion limit of the membranes used being molecules with a molecular weight of more than 10 kDa. These molecules are found almost exclusively in the retentate (retentate). It is obvious to the person skilled in the art that the development of new separation techniques and new combinations of process steps will always make it possible to achieve an improvement in the purification process. This is directly advantageous with regard to the yield and thus the production costs.

The invention therefore relates to DNA molecules of the following form (alternative terms: expression cassettes):

P_x-S_x-B_n- (ZR) -transport peptide- (Z)₁Z₂) -protein (Y) - (Z)₁Z₂) -protein (Y)_m)-T；

The expression cassette encodes a protein in which the encoded transit peptide passes through the sequence Z₁Z₂Is linked to a second protein which in turn is linked via Z₁Z₂Linked to protein Y1, Y1 either corresponds to Y or is different from Y, and the transport peptide increases Y and/or Y_mIn the DNA molecule:

P_xany promoter DNA sequence selected in such a way that an optimal yield of the protein of interest is obtained;

S_xis the corresponding coding DNA of any signal or leader sequence which allows optimal yields;

B_nis a codon or a chemical bond of 1-15 genetically encodable amino acids;

z is the codon of an amino acid selected from Lys and Arg;

Z₁is a codon for an amino acid selected from Lys and Arg;

Z₂is a codon for an amino acid selected from Lys and Arg;

r is Arg codon;

transit peptides are DNA sequences which encode peptides which are efficiently transported and which are capable of crossing the cell membrane, such as hirudin or hirudin derivatives;

protein Y is a DNA sequence encoding any protein that can be produced and secreted by yeast;

protein Y_mIs a DNA sequence (m-1-5) or a chemical bond (m-0) encoding any protein that can be produced and secreted by yeast;

t is an untranslated DNA sequence that is advantageous for expression.

Another embodiment of the invention is a fusion protein encoded by any of the DNA molecules described above.

Another embodiment of the invention are multicopy vectors and plasmids containing the DNA molecules described above.

Another embodiment of the invention is a host cell containing the above-described DNA molecule or the above-described multicopy vector or plasmid as part of a chromosome, as part of a minichromosome, or extrachromosomally, wherein the host cell is preferably a yeast cell, in particular a yeast cell selected from the group consisting of saccharomyces cerevisiae (s.

Another embodiment of the present invention is a process for the fermentation of the above-mentioned proteins, wherein

(a) Allowing the above DNA molecule, multicopy vector or plasmid to express in the above host cell and

(b) isolating the expressed protein from the supernatant of the cell culture,

wherein the pH is adjusted to 2.5-3.5 in order to precipitate unwanted proteins, in particular after the fermentation has been completed, and the expressed proteins are then separated from the supernatant of the precipitate.

Another embodiment of the present invention is the above process, wherein after separating the fermentation supernatant from the host cells, the host cells are re-cultured in fresh medium and the released fusion protein is separated from the supernatant obtained from each culture.

Another embodiment of the present invention is the above method, wherein the step for concentrating the expressed protein in the supernatant after the precipitation is selected from the group consisting of microfiltration, hydrophobic interaction chromatography and ion exchange chromatography.

Another embodiment of the present invention is a process for the preparation of insulin, wherein

(a) Expressing proinsulin in the above method as protein (Y) of the above expression cassette;

(b) isolating the proinsulin from step (a) and treating with trypsin and carboxypeptidase B; and is

(c) Isolating insulin from the reaction mixture of step (b),

in particular, wherein the transit peptide is hirudin or a hirudin derivative which is disrupted or the biological activity of which is lost after step (a) or (b).

Another embodiment of the invention is a protein which is a hirudin derivative having two basic amino acid residues at the C-terminus.

Leeches of the leech species (Hirudo) have formed, for example, various isoforms of the thrombin inhibitor hirudin. Hirudins have been optimized to meet pharmaceutical requirements by artificial modification, e.g.by changing the N-terminal amino acid (e.g.EP 0324712). The invention includes the use of hirudin and hirudin variants. Particular embodiments of the present invention employ a natural hirudin isoform (all of which are collectively referred to as "hirudin"). Naturally occurring isoforms are, for example, Val-Val-hirudin or Ile-Thr-hirudin. Other embodiments of the invention use variants of the natural hirudin isoforms. Variants are derived from the natural hirudin isoforms but contain, for example, additional amino acids and/or amino acid deletions and/or amino acid changes compared to the natural isoforms. The hirudin variants may contain alternating peptide fragments of the natural hirudin isoform and new amino acids. Hirudin variants are known and described, for example, in DE 3430556. Hirudin variants are commercially available in the form of proteins (Calbiochem Biochemicals, cat. No.377-853, -950, -960). The term "hirudin derivative" refers to a sequence which is at least 40% homologous to natural hirudin.

The expression cassette is preferably introduced into a yeast such as Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha or Pichia pastoris. The expression cassette described above may be stably integrated in one or more copies into the genome of the particular yeast or may be present in a multicopy vector extrachromosomally. It will be apparent to those skilled in the art that the technique is also applicable to other systems such as animal cell cultures and plant cells. This is also a subject of the present invention.

The expression systems described below serve as examples. It will be apparent to those skilled in the art that in order to introduce an expression cassette into the system selected above, an appropriate recombinant DNA construct must be made, depending on the type of host system selected. Thus, industrial fermentations can be optimized according to the host/vector system chosen. The following examples are, therefore, not to be taken in a limiting sense.

Example 1: construction of a Yeast expression plasmid encoding hirudin (Refludan) -Lys-Arg-Mini-proinsulin

The starting materials were plasmids pK152(PCT/EP00/08537), pSW3 (EP-A0347781) and recombinant yeast plasmid derivatives encoding bovine interleukin 2 (Price et al Gene 55, 1987). This yeast plasmid can be distinguished by the fact that it carries the alpha-factor leader sequence under the control of the yeast ADH2 promoter. This sequence is followed by a KpnI restriction enzyme recognition site to which the bovine interleukin 2cDNA sequence, which after manipulation contains an NcoI restriction enzyme recognition site at the untranslated 3' end, which is unique within the vector. Thus, this cDNA sequence can be easily removed from the plasmid by KpnI/NcoI cleavage. Since the reported expression yields are good, the remaining 3' interleukin 2 sequence (like T) is considered to have mRNA stabilizing effectIn use, it need not be deleted or replaced by a yeast termination sequence. Plasmid pK152 carries the DNA sequence coding for Leu-hirudin (Refludan) and plasmid pSW3 carries the DNA sequence coding for mini-proinsulin. The gene sequence coding for hirudin-LysArg-miniproinsulin was first prepared by PCR techniques. For this purpose, Expedite is used^TmThe DNA synthesis system prepared 4 primers.

Hir _ insfkr (SEQ ID NO: 1, encoded protein fragment: SEQ ID NO: 2)

I P E E Y L Q K R F V N Q H L C

5′-ATCCCTGAGGAATACCTTCAGAAGCGATTTGTTAACCAACACTTGTGTGG-3`

59 60 61 62 63 64 65 B1 B2 B3 B4 B5 B6 B7

ii.hir_insrevkr(SEQ ID NO：3)

5′-CCTCACAAGTG TTGGTTAACA AATCGCTTCT GAAGGTATTC CTCAGGGAT-3`

iii hirf1(SEQ ID NO: 4, encoded protein fragment: SEQ ID NO: 5)

L T Y T D C

5′-TTTTTTTGGATCCTTTGGATAAAAGACTTACGTATACTGACTGCAC

iv.insnco1rev(SEQ ID NO：6)

5′-TTTTTTCCAT GGGTCGACTATCAG

Primer hir _ insfkr describes the ligation between the codon for the terminal amino acid of hirudin (59-65) and the insulin sequence B1-B7 via the Lys-Arg linker. Primer hir _ instrevkr is its 100% complement. The primer hirf1 encodes the initial part of the hirudin gene which extends into the KpnI cleavage site (see description in EP-A0324712).

Primer insco 1rev shows the 3' end of the synthetic mini-proinsulin according to EP-A0347781. Two standard polymerase chain reactions were performed with the primer pair hirf1/hir _ insrevkr and the DNA of plasmid pK152 as template and the primer pair hir _ insfkr/insnco1rev and the DNA of plasmid pSW3 as template. The reaction was carried out in 100. mu.l of PCR buffer, in each case 200nmol of primer, 1. mu.l of polymerase and 100ng of vector. Step 1 was an incubation at 95 ℃ for 2 minutes. Then 25 cycles of 95 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 30 seconds were followed. After the last cycle, the reaction was incubated at 72 ℃ for 3 minutes and then stopped.

Due to 100% complementarity of the primers hir _ instrevkr and hir _ instfkr, the two DNA products overlap according to the above sequence, so that in the third reaction, the DNA fragment generated using the products of the first two reactions as template and using primers hirf1 and instco 1rev encodes hirudin and miniproinsulin separated by Lys-Arg. The PCR fragment was digested with the enzymes KpnI and NcoI and inserted into the p.alpha.ADH 2 vector cut with KpnI 1/NcoI by T4 ligase reaction. Similarly to example 7 of EP-A0347781, the competent E.coli strain MM294 was transformed with the ligation mixture. Plasmid DNA was then isolated from both clones and characterized by DNA sequence analysis. After the inserted DNA sequence was determined, plasmid DNA preparations were used to transform bakers yeast strain Y79 cells according to the examples. However, unlike the examples, when the p α ADH2 vector was used, the selection for complementation of the trp1-1 mutation was performed after the vector was introduced. For another control, plasmid DNA was re-isolated from yeast transformants and analyzed by restriction analysis. The constructed expression vector is denoted pADH2Hir _ KR _ Ins. Expression was performed according to example 4.

Example 2: construction of Yeast expression plasmids encoding hirudin (Refludan) -Lys-Arg-insulin B chain-Lys-Arg-insulin A chain

The proinsulin derivative described in patent application EP-A0195691 may contain a dipeptide XY, where X and Y each correspond to Lys or Arg, as a linker for the B and A chains of insulin. The following examples describe the preparation of expression vectors for such proinsulin derivatives. For example, a DNA sequence encoding a proinsulin derivative in the B chain-Lys-Arg-A chain form is selected and synthesized accordingly.

Synthesis of gene fragments was carried out according to example 1. The oligonucleotide sequences used were hirf1 and insnco1 rev. Oligonucleotides B _ KR _ Af1 and B _ KR _ Arev1 were synthesized de novo.

The sequence of B _ KR _ Af1 is (SEQ ID NO: 7)

5′CTTCTACACTCCCGCGCGG-3`

The sequence of B _ KR _ Arev1 is (SEQ ID NO: 8)

5′CAACATTGTTCAACGCCGCGTTTCGTCTTT-3`

The portions of the two primers shown in bold show partially overlapping sequences. Both primers were precisely paired with the mini-proinsulin sequence in EP-A0347781, except for the 6 underlined nucleotides. The underlined parts correspond to the Lys and Arg codons. The DNA of plasmid pADH2Hir _ KR _ Ins constructed according to example 1 was used as template in PCR.

Two polymerase chain reactions were performed with the primer pairs hirf1/B _ KR _ Arev and insnco1rev/B _ KR _ Af1 as described in example 1. The template in each reaction was the plasmid pADH2Hir _ KR _ Ins DNA constructed in example 1. In the third PCR, the primer pairs were hirf1 and insnco1, with the products of the first two reactions as templates. The reaction product of the third PCR was cut with NcoI/Sall and inserted into the opened p α ADH2 vector. After sequence and restriction analysis, the correct plasmid was called pADHHirKR _ B _ KR _ A.

Example 3: construction of a Yeast plasmid encoding hirudin-Lys-Arg-Simian proinsulin

Patent application EP-A489780 describes a plasmid: pINT90d, which contains cDNA of simian proinsulin (Wetekam et al, Gene 19, p.179-183, 1982). The plasmid and the DNA of plasmid pK152 were used as templates. The primer hirf1 described in example 1 was used and three additional primers were synthesized.

Primer insncorev binds to the 3' region of the insulin gene cloned in pINT90d in reverse and has the sequence:

5′-TTTTTTCCATGGTCATGTTTGACAGCTTATCAT-3`(SEQID NO：9)

the underlined sequence indicates the recognition site for the restriction enzyme NcoI.

The primer hir _ inspkr sequence is:

5′-ATCCCTGAGGCCTTCA GTTT GTGAACCAGC ACCTGTGCGG C-3`(SEQ ID NO：10)

the Lys-Arg linker between hirudin and proinsulin is indicated here by the nucleotides shown in bold.

Primer hir _ instrevkr is fully complementary to primer hir _ instkr, and has the sequence:

5′-GCCGCACAGG TGCTGGTTCA CAAACTGAAGGTAT TCCTCAGGGA T-3`(SEQ ID NO：11)

in analogy to example 1, two polymerase chain reactions were carried out. The primer pair hirf1/hir _ insrevkr and plasmid pK152 DNA reacted, and the primer pair hir _ insfkr/insncorev and plasmid pINT90d DNA reacted. The products of both reactions were used as templates for the third PCR, with the primer pair hirf1/insncorev as described in example 1. The DNA product of this reaction includes the sequence of hirudin-Lys-Arg-proinsulin. This was then cleaved with the enzymes NcoI and KpnI and, analogously to example 1, inserted into the plasmid p α ADH 2. Thus, any expression vector for a native proinsulin derivative can be constructed.

Example 4: expression of the recombinant product

Expression is divided into two phases. First, preculture was performed in a yeast minimal medium. The following ingredients were contained per liter of medium:

6.7 g-Yeast Nitrogen Source (Yeast Nitrogen base) (without amino acids)

5.0 g-Casein amino acid (No vitamin)

0.008% -adenine

0.008% -uracil

2% -glucose

The main medium or expression medium is inoculated with a pre-culture sample.

The main culture medium contains per liter:

10 g-Yeast extract

20 g-peptone

0.008% -adenine

0.008% -uracil

4% -glucose

The above medium was used for expression in shake flasks by the following method: 0.3ml of the overnight cultured preculture was diluted with 80ml of the preheated medium and incubated at 30 ℃ for about 24 hours with vigorous shaking. In each case, 1 ml of the culture produced in this way was centrifuged after determination of the optical density, the cells were removed and the supernatant was lyophilized and analyzed by SDS-PAGE. The content of biologically active hirudin is determined by a thrombin inhibition assay.

An alternative fermentation protocol is for cells removed by filtration or careful centrifugation. While isolating the protein of interest from the medium, the removed cells are placed in a fresh pre-heated main medium containing alcohol and not more than 0.5% glucose as carbon source, so that the fermentation can be continued without interruption. This step can be repeated up to 5 times.

Example 5: thrombin inhibition assay

The concentration of hirudin is determined according to the method of Grie β bach et al (Thrombosils Research 37, pp.347-350, 1985). To this end, a specific amount of a Refludan standard is included in the assay to establish a calibration curve from which the yield in mg/l can be directly determined.

Example 6: cloning and expression of hirudin-Lys-Arg-mini-proinsulin fusion protein in Pichia pastoris system

The cloning and expression kit for preparing recombinant protein by using pichia pastoris as a host system is sold. To this end, detailed technical solutions for the preparation and subsequent expression of the pichia pastoris system for the production of the desired recombinant protein are provided, and thus, when using the solutions, only the construction of an expression vector encoding the desired protein will be described. Easyselect was used^TMPichia expression kit (Cat. No. K1740-01).

The pPICZ α a vector is part of the kit. Opening the vector by restriction enzymes XhoI and SacII makes it possible to add the protein of interest to the α -factor leader sequence and detect secretion of the protein into the supernatant similarly to example 1. Two primers are required for cloning. The sequence of primer pichia _ H _ If1(SEQ ID NO: 12) is:

5′-TTTTTTTCTCGAGAAAAGA CTTACGTATACTGAC-3′

Xhol Hir₁ Hir₂etc. of

The sequence of primer pichia _ H _ Irev2(SEQ ID NO: 13) is:

5′-TTTTTGGCGCCGAATTCACTATTAGTTACAGTAGTTTTCC-3′

SacII EcoRI A21

the template used was the DNA of plasmid pADH2Hir _ KR _ Ins. Standard PCR with these two primers yielded a DNA product containing the hirudin-Lys-Arg-miniproinsulin sequence extended by the XhoI and SacII integration sites. After appropriate cleavage of the DNA product and isolation of the fragments, the fragments can be inserted into the opened vector DNA by T4DNA ligase reaction. In a slight variation from the manufacturer's protocol, E.coli strain MM294 described in example 1 was transformed by the ligation mix and recombinant colonies were screened by zeocin selection plates. Plasmid DNA was re-isolated from the clones and then identified by restriction and DNA sequence analysis. The plasmid constructed in this way was used to prepare a Pichia pastoris expression clone for the production of the peptide according to the manufacturer's protocol.

Example 7: purification of mini-proinsulin and hirudin

Purification requires the separation of the two proteins at an early stage. After expression was complete, the medium was analyzed by analytical RP-HPLC. Unlike most other polypeptides which occur in the supernatant as a result of spontaneous lysis or secretion by yeast cells, these two proteins, hirudin and miniproinsulin, do not precipitate at pH 2.5-3. The medium was therefore mildly acidified and then the pellet and cells were removed by centrifugation after the pellet was complete. After centrifugation, the medium is adjusted to pH 3.5-7 and chromatographed by hydrophobic interaction, for example by using a cell containing DiaionLayer of materialThe column separates the two fractions hirudin and miniproinsulin from each other. Hirudin can then be separated from the hirudin-containing fraction according to EP-A0549915 and insulin can be separated from the proinsulin-containing fraction according to EP-A0347781.

Example 8: preparation of insulin from mini-proinsulin

At the end of the expression phase, the medium is adjusted to pH 6.8 and trypsin is added with stirring to a final concentration of 4-8 mg/l. After an incubation time of about 4 hours, the fermentation broth treated in this way is adjusted to a pH of 2.5-3. After 1-6 hours of precipitation, the precipitate was removed. Then by ion exchange chromatography, for example in a buffer (pH3.5) containing 50mM lactic acid and 30% isopropanolThe resulting mono-Arg-insulin is isolated by chromatography. The elution was carried out using a NaCl gradient of 0.05-0.5M. The product-containing fractions are diluted 1: 1 with water and ZnCl is added₂To form 0.1% strength ZnCl₂And (3) solution. mono-Arg-insulin precipitates at pH 6.8 and can then be converted into insulin, for example, according to EP-a 0324712.

Example 9: preparation of insulin from proinsulin after filtration

At the end of the expression phase, the cells and supernatant components are removed by precipitation at pH 2.5 to 3. The medium was then concentrated by filtration through a membrane with an exclusion limit of 10 kDa. Similar to hirudin derivatives, mini-proinsulin is present in large amounts in the retentate, which can then be processed to insulin according to example 8.

Sequence listing

<110> Anduwan Teddy medicine Germany Co., Ltd

<120> hypersecretoble peptides, methods for their preparation and parallel enhancement of the secretory form of one or more other polypeptides

<130>DEAV2001/0007

<140>10108100.6

<141>2001-02-20

<160>13

<170>Patent In Ver.2.1

<210>1

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: hir _ inspkr

<400>1

atccctgagg aataccttca gaagcgattt gttaaccaac acttgtgtgg 50

<210>2

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: protein hir _ inspkr

<400>2

Ile Pro Glu Glu Tyr Leu Gln Lys Arg Phe Val Asn Gln His Leu Cys

1 5 10 15

<210>3

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: hir _ instrevkr

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cctcacaagt gttggttaac aaatcgcttc tgaaggtatt cctcagggat 50

<210>4

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: hirf1

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tttttttgga tcctttggat aaaagactta cgtatactga ctgcac 46

<210>5

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: protein hirf1

<400>5

Leu Thr Tyr Thr Asp Cys

1 5

<210>6

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: instcholerev

<400>6

ttttttccat gggtcgacta tcag 24

<210>7

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: b _ KR _ Af1

<400>7

cttctacact ccaaagacga aacgcggtat cg 32

<210>8

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: b _ KR _ Arev1

<400>8

caacattgtt caacgatacc gcgtttcgtc ttt 33

<210>9

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: insncorev

<400>9

ttttttccat ggtcatgttt gacagcttat cat 33

<210>10

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: hir _ inspkr

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atccctgagg aataccttca gaagcgattt gtgaaccagc acctgtgcgg c 51

<210>11

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: hir _ instrevkr

<400>11

gccgcacagg tgctggttca caaatcgctt ctgaaggtat tcctcaggga t 51

<210>12

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: pichia _ H _1f1

<400>12

tttttttctc gagaaaagac ttacgtatac tgac 34

<210>13

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: pichia _ H _ lrev2

<400>13

tttttggcgc cgaattcact attagttaca gtagttttcc 40

Claims (15)

1. A DNA molecule of the form:

P_x-S_x-B_n- (ZR) -transport peptide- (Z)₁Z₂) -protein (Y) - (Z)₁Z₂) -protein (Y)_m)-T；

The DNA molecule encodes a protein in which the transport peptide encoded therein passes through the sequence Z₁Z₂Is linked to a second protein which in turn is linked via Z₁Z₂Linked to protein Y1, Y1 either corresponds to Y or may be different from Y, and the transport peptide increases Y and/or Y_mIn the D NA molecule:

P_xany promoter DNA sequence selected in such a way that an optimal yield of the protein of interest is obtained;

S_xis the corresponding coding DNA of any signal or leader sequence which allows optimal yields;

B_nis a codon or a chemical bond of 1-15 genetically encodable amino acids;

z is the codon of an amino acid selected from Lys and Arg;

Z₁is a codon for an amino acid selected from Lys and Arg;

Z₂is a codon for an amino acid selected from Lys and Arg;

protein Y_mIs a DNA sequence encoding proinsulin or a derivative thereof capable of being produced and secreted by yeast, wherein m is 0-5; when m is 0, Y_mIs a chemical bond;

r is an arginine codon;

the transit peptide is a DNA sequence encoding hirudin or a hirudin derivative;

protein Y is a DNA sequence encoding proinsulin or a derivative thereof which is capable of being produced and secreted by yeast, and when Y is_mNot a chemical bond, the biological activity of protein Y is not impaired by the extension of the basic dipeptide, or it allows degradation of this extended portion by carboxypeptidases;

t is an untranslated DNA sequence that is advantageous for expression.

2. The DNA molecule according to claim 1, wherein the protein Y is a DNA sequence encoding proinsulin, insulin A chain, or insulin B chain; protein Y_mIs a DNA sequence encoding mini-proinsulin, insulin A chain, or insulin B chain; or Y_mIs a chemical bond.

3. A protein encoded by a DNA molecule according to claim 1 or 2.

4. A multicopy vector comprising the DNA molecule of claim 1 or 2.

5. A plasmid comprising the DNA molecule of claim 1 or 2.

6. A host cell comprising the DNA molecule of claim 1 or 2, the multicopy vector of claim 4 and/or the plasmid of claim 5 as part of a chromosome, as part of a minichromosome or in extrachromosomal form.

7. The host cell according to claim 6, wherein said host cell is a yeast.

8. The host cell according to claim 7, which is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Hansenula polymorpha and Pichia pastoris.

9. A process for the fermentation of a protein according to claim 3, wherein

(a) Expressing the DNA molecule of claim 1 or 2, the multicopy vector of claim 4 or the plasmid of claim 5 in a host cell according to any one of claims 6 to 8; and is

(b) The expressed protein is isolated from the cell culture supernatant.

10. The process according to claim 9, wherein after fermentation is complete, the pH is adjusted to 2.5-3.5 to precipitate unwanted proteins and the expressed proteins are separated from the supernatant after precipitation.

11. The method according to claim 10, wherein after separation of the fermentation supernatant from the host cells, the host cells are repeatedly cultured with fresh medium and the released fusion protein is separated from the supernatant obtained in each cultivation step.

12. The method according to any one of claims 9 to 11, wherein the step of separating the expressed protein from the supernatant of the cell culture is selected from the group consisting of microfiltration, hydrophobic interaction chromatography and ion exchange chromatography.

13. A process for the preparation of insulin, wherein

(a) Expressing proinsulin by a method according to claim 9 or 10, wherein the proinsulin is expressed in the form of the protein (Y) in the DNA molecule of claim 1,

(b) separating the proinsulin from step (a) and treating with trypsin and carboxypeptidase B, and

(c) isolating insulin from the reaction mixture of step (b).

14. The method according to claim 13, wherein hirudin or a hirudin derivative as transport peptide is destroyed or the biological activity is lost after step (a) or (b).

15. A protein according to claim 3, wherein said protein is a hirudin derivative having two basic amino acid residues at the C-terminus.