HK1124341B - Cleavage of precursors of insulins by a variant of trypsin - Google Patents

Description

Cleavage of insulin precursors by trypsin variants

The present invention relates to the production of recombinant trypsin variants with increased substrate specificity for arginine compared to lysine in non-animal host organisms. Furthermore, the invention relates to recombinant trypsin and the production thereof. The invention also provides the use of recombinant porcine pancreatic trypsin variants for cleaving insulin precursors, and kits comprising the trypsin variants.

Trypsin is a serine protease that catalyzes The hydrolytic cleavage of peptides at The carboxy terminus of The basic amino acids arginine and lysine (Keil b., (1971). The Enzyme, vol.2, third edition, editors, Boyer, acad. The recombinant porcine pancreatic trypsin has a molecular weight of about 23000 daltons and has optimal enzymatic activity at ph 8.0.

Trypsin is used in industrial processes for the production of insulin and insulin analogues. The production of these biomolecules is described in the literature and several methods have been chosen. Chemical synthesis of human insulin and insulin analogues is not feasible from an economic point of view. Thus, there are now mainly two methods of producing insulin and insulin analogs, namely a semi-synthetic method using porcine insulin as a starting material, and the use of genetically modified microorganisms for the expression of recombinant insulin.

Insulin is a 51 amino acid polypeptide that is divided into 2 amino acid chains: an A chain having 21 amino acids and a B chain having 30 amino acids. The chains are linked to each other by 2 disulfide bonds. Insulin preparations have been used for many years in the treatment of diabetes. Not only naturally occurring insulin but also insulin derivatives and analogues have recently been used.

Insulin analogs are analogs of naturally occurring insulin (i.e., human or animal insulin) that differ by the substitution of at least one naturally occurring amino acid residue with another amino acid residue and/or the addition/removal of at least one amino acid residue (otherwise identical) from the corresponding naturally occurring insulin. The added and/or substituted amino acid residues may also be non-naturally occurring amino acid residues.

An insulin derivative is a derivative of a naturally occurring insulin or insulin analog that results from a chemical modification. Chemical modifications may be in the addition of one or more specific chemical groups to one or more amino acids, for example.

Examples of insulin derivatives are B29-N-myristoyl-des (B30) human insulin, B29-N-palmitoyl-des (B30) human insulin, B29-N-myristoyl human insulin, B29-N-palmitoyl human insulin, B28-N-myristoyl Lys^B28Pro^B29Human insulin, B28-N-palmitoyl-Lys^B28Pro^B29Human insulin, B30-N-myristoyl-Thr^B29Lys^B30Human insulin, B30-N-palmitoyl-Thr^B29Lys^B30Human insulin, B29-N- (N-palmitoyl-Y-glutamyl) -des (B30) human insulin, B29-N- (N-lithocholyl)(liticholol) -Y-glutamyl) -des (B30) human insulin, B29-N- (. omega. -carboxyheptadecanoyl) -des (B30) human insulin and B29-N- (. omega. -carboxyheptadecanoyl) human insulin.

Insulin analogues are described in EP 0214826, EP 0375437 and EP 0678522. EP 0214826 relates to substitutions of B27 and B28, etc. EP 0678522 describes insulin analogues with a number of amino acids, preferably proline, but no glutamic acid at position B29.

The other insulin analogue is Lys^B28Pro^B29Human insulin, B28Asp human insulin, human insulin wherein the proline at position B28 has been substituted by Asp, Lys, Leu, Val or Ala and the Lys at position B29 may be substituted by Pro; AlaB26 human insulin; des (B28-B30) human insulin; des (B27) human insulin or des (B30) human insulin.

EP 0375437 includes insulin analogs with lysine or arginine at position B28, which may optionally additionally be modified at B3 and/or a 21.

In EP 0419504 insulin analogues are disclosed which are protected against chemical modifications, wherein asparagine at position B3 and at least one amino acid at position a5, a15, a18 or a21 are modified. In WO 92/00321 insulin analogues are described in which at least one of the amino acids in positions B1-B6 is replaced by lysine or arginine.

Important insulin analogues described herein are also "insulin glargine" (Gly a (21), Arg B (31), Arg B (32) human insulin) and "insulin glulisine" (Lys B (3), Glu B (29) human insulin).

Recombinant DNA methods allow precursors of insulin or insulin analogs, in particular human proinsulin or proinsulin having an amino acid sequence and/or amino acid chain length different from human insulin to be prepared in microorganisms. Proinsulin prepared from genetically modified E.coli (Escherichia coli) cells did not have any correctly bound cystine bonds. The process for obtaining human insulin using E.coli (EP 0055945) is based on the following process steps:

fermentation of the microorganism, cell separation, cell disruption, isolation of insulin or insulin analogues, refolding by formation of the respective disulfide bonds into the desired (native) three-dimensional structure to produce pre-pro-insulin ("PPI"), trypsin cleavage of the respective pre-pro-insulin (possibly in the presence of carboxypeptidase B), basic purification, a first chromatography step, final enzymatic cleavage to produce human insulin or the respective insulin analogue, a second chromatography step and final purification by HPLC, crystallization and drying.

Trypsin cleavage of pre-proinsulin is an enzymatic and complex reaction: the pro sequence and C-peptide are cleaved in this step to yield the respective products. For example, in the case of human insulin production, the desired values are Arg (B31), Arg (B32) -insulin and Arg (B31) -insulin (DE 19821866).

However, trypsin cleavage results in the formation of many by-products as a result of unwanted side reactions. Trypsin is an endoprotease (serine type) that cleaves peptide bonds at the C-terminal arginine (Arg) or lysine (Lys) residues. The trypsin cleavage of the pre-proinsulin molecule can occur simultaneously at different cleavage sites. Due to the many cleavage edges in a particular pre-proinsulin molecule, many unwanted byproducts can be formed during the trypsin cleavage reaction. As can be seen in fig. 1, many of the by-products generated during the cleavage reaction are the result of peptide bond cleavage at the C-terminal side of Lys rather than Arg residues.

For all pre-proinsulin, the cleavage site between the pre-sequence and the insulin B chain is monobasic. At this junction, only one cleavage reaction can occur.

There are different cleavage sites at the two other junctions-B-chain/C-peptide and C-peptide/A-chain. In the case of human and insulin glargine, the cleavage site between B-chain/C-peptide and C-peptide/A-chain is dibasic (Arg-Arg and Lys-Arg, respectively). In addition, cleavage after B29-Lys results in the formation of B30-des-Thr ("des-Thr").

For human insulin, only trypsin cleavage after residues B31-Arg and B32-Arg yields products of value for the B-chain/C-peptide junction, namely B31-Arg insulin ("mono-Arg") and B31-Arg, B32-Arg insulin ("di-Arg"). These products can be summarized as "Arg-insulin". Cleavage after B32-Arg is important in the production of insulin glargine, since only di-Arg-insulin can be used. For human insulin and insulin glargine, cleavage after B29-Lys results in des-Thr formation. For insulin glargine, this cleavage site is monobasic and the possible product is an Arg-containing species.

For the C-peptide/A-chain, trypsin cleavage after Arg rather than Lys residues is important for the production of valuable products. The erroneous cleavage after Lys leads to the formation of a0 by-product.

To overcome the disadvantages of the art, it is desirable to use trypsin during the pre-proinsulin cleavage, which has enhanced Arg specificity for the different cleavage sites exemplified in FIG. 1. By increasing the Arg-specificity of trypsin, and thus by decreasing the Lys-specificity, one can expect a decrease in the formation of by-products, in particular des-Thr and A0-components. Sichler et al, FEBSLett. (2002) 530: 220-224 examined the effect of amino acid exchange at position 190 in human trypsin (chymotrypsinogen numbering according to Huber, R. & Bode, W., Acc. chem. Res. (1978) 11: 114-122). When an artificial substrate was used, it was found that the change from wild-type serine to mutated alanine at this position resulted in an increase in the selectivity of the cleavage site for arginine and a decrease in lysine. Meanwhile, the enzymatic activity of the mutant was found to be reduced by about 1/2 compared to the wild type. Testing of recombinant human wild-type trypsin and human trypsin mutants (amino acid change from serine to alanine at position 190) for pre-proinsulin treatment resulted in the formation of a large number of byproducts for both enzymes, indicating that an increase in Arg-selectivity is not assignable to cleavage of pre-proinsulin (see below, example 1).

In view of the state of the art, the problem to be solved is to provide variants of trypsin that exhibit increased cleavage site selectivity for arginine without a large loss of proteolytic activity. Another specific problem to be solved is to provide a variant of trypsin with increased cleavage site selectivity for arginine residues in the cleavage site for pre-proinsulin processing, as illustrated in fig. 1.

Thus, this problem is solved by providing a variant of porcine trypsin having an amino acid change from serine to alanine at position 172. In a preferred embodiment, the Ser172Ala variant of porcine trypsin is provided by recombinant means. Surprisingly, it was found that the enzymatic activity of the Ser172Ala variant of porcine trypsin for the pre-proinsulin cleavage reaction was almost equal to the wild-type enzyme.

The serine at amino acid position 190 detected in human trypsin by Sichler et al (FEBS Lett. (2002) 530: 220-224) corresponds to the amino acid sequence shown in SEQ ID NO: 1, serine 172 of porcine trypsin provided in (1). Both positions are part of the so-called S1 site of the trypsin-like serine protease.

One embodiment of the invention is therefore the use of Ser172Ala porcine trypsin in the preparation of insulin, insulin analogues or insulin derivatives.

Another embodiment of the invention is a process for the preparation of insulin, an insulin analogue or an insulin derivative, wherein

(a) Pre-pro-insulin, pre-pro-insulin analogue or pre-pro-insulin derivative was cleaved with Ser172Ala porcine trypsin,

(b) the resulting cleavage product is isolated and

(aa) if one of the cleavage products obtained is an insulin analogue or an insulin derivative, obtaining the insulin analogue or insulin derivative, or

(bb) further processing those cleavage products which are precursors of insulin, insulin analogues or insulin derivatives, and isolating and obtaining the insulin, insulin analogues or insulin derivatives obtained from this further processing;

wherein the insulin is preferably human insulin; and the insulin analogue is selected from Lys^B28Pro^B29Human insulin, B28Asp human insulin, human insulin (wherein proline at position B28 has been substituted by Asp, Lys, Leu, Val or Ala and wherein Lys at position B29 may be substituted by Pro); AlaB26 human insulin; des (B28-B30) human insulin; des (B27) human insulin and des (B30) human insulin, and the insulin derivative is selected from the group consisting of: B29-N-myristoyl-des (B30) human insulin, B29-N-palmitoyl-des (B30) human insulin, B29-N-myristoyl human insulin, B29-N-palmitoyl human insulin, B28-N-myristoyl Lys^B28Pro^B29Human insulin, B28-N-palmitoyl-Lys^B28Pro^B29Human insulin, B30-N-myristoyl-Thr^B29Lys^B30Human insulin, B30-N-palmitoyl-Thr^B29Lys^B30Human insulin, B29-N- (N-palmitoyl-Y-glutamyl) -des (B30) human insulin, B29-N- (lithocholyl) -Y-glutamyl) -des (B30) human insulin, B29-N- (omega-carboxyheptadecanoyl) -des (B30) human insulin and B29-N- (omega-carboxyheptadecanoyl) human insulin.

In a preferred embodiment of the invention the insulin analogue is insulin glulisine or insulin glargine.

In another embodiment of the invention, the further processing of those cleavage products mentioned (being precursors of insulin, insulin analogues or insulin derivatives) comprises cleavage of said products with carboxypeptidase B, with the exception of insulin glargine.

In another embodiment of the invention, the cleavage with Ser172Ala porcine trypsin is carried out at a pH in the range of 7.5 to 9.5 (preferably 8.3), at a temperature between 1 ℃ and 30 ℃, more preferably between 8 and 15 ℃, most preferably at 8 ℃; and the enzymatic reaction is terminated by acidifying the sample, preferably by adding 1N or 2N HCl solution.

Another embodiment of the invention is a polypeptide consisting of the sequence SEQ ID NO: 3, preferably a Ser172Ala porcine trypsin characterized by the sequence SEQ ID NO: 4DNA encoding Ser172Ala porcine trypsin.

Another embodiment of the invention is a polypeptide consisting of SEQ ID NO: 6 DNA encoding Ser172Ala pre-trypsinogen. The signal peptide of this pre-trypsinogen is derived from the alpha factor of Saccharomyces cerevisiae (Saccharomyces cerevisiae).

Another embodiment of the present invention is a vector comprising the above DNA.

Another embodiment of the invention is a method of producing Ser172Ala porcine trypsin comprising the steps of:

(a) providing a carrier according to claim 16, wherein said carrier is,

(b) the carrier is used for transforming a microbial host strain,

(c) cultivating the transformed microbial host strain in a growth medium comprising nutrients, whereby the microbial host strain expresses Ser172Ala porcine trypsin or Ser172Ala porcine trypsinogen,

(d) if the expression product of (c) is Ser172Ala porcine trypsinogen, converting it into mature Ser172Ala porcine trypsin, and

(e) purification of Ser172Ala porcine trypsin from a microbial host strain and/or growth medium,

in particular wherein the microbial host strain is a methylotrophic yeast strain selected from the species Hansenula (Hansenula), Pichia (Pichia), Candida (Candida) and Torulopsis (Torulopsis); preferably wherein the microbial host strain is selected from the group consisting of Pichia pastoris (Pichia pastoris), Hansenula polymorpha (Hansenula polymorpha), Candida boidinii (Candida boidinii) and Torulopsis glabrata (Torulopsis glabrata).

In this document, the terms "variant of porcine trypsin" and "variant of porcine trypsin" refer to the protein as a variant, i.e. the allelic form of mature porcine pancreatic trypsin protein isoform I, as defined by the amino acid sequence according to SEQ ID NO: 1, amino acid substitution at position 172.

For shorthand designations of porcine trypsin variants described herein, it is noted that the numbers refer to the amino acid sequences as set forth in SEQ ID NO: 1 amino acid residue/position of the amino acid sequence of mature porcine pancreatic trypsin as given. Amino acid identification uses the three letter abbreviations and one letter of the amino acids, namely Asp D aspartic acid, Ile I isoleucine, Thr T threonine, Leu L leucine, Ser S serine, Tyr Y tyrosine, Glu E glutamic acid, Phe F phenylalanine, Pro P proline, His H histidine, Gly G glycine, Lys K lysine, Ala A alanine, Arg R arginine, Cys C cysteine, Trp W tryptophan, Val V valine, Gln Q glutamine, Met M methionine, Asn N asparagine. The amino acids at specific positions of the amino acid sequence are given by their three-letter abbreviations and numbers. Thus, Ser172 is referred to in SEQ ID NO: 1, serine residue at amino acid position 172. Substitutions of different amino acids are given as the addition of a three letter abbreviation after the number indicating position. For example, "Ser 172 Ala" refers to SEQ ID NO: 3 Ser at position 172 by Ala.

The term "increased cleavage site selectivity" of a trypsin variant refers to a shift in specificity for hydrolytic cleavage, thus for the variant, the shift results in a preferred cleavage at the carboxyl group of arginine rather than lysine.

When quantifying trypsin activity, this document refers to "units (U)". The proteolytic activity of trypsin and its variants was quantified using photometric assays (using Chromozym TRY, Chromozym TH and Chromozym PL (Roche Diagnostics GmbH) as substrates). The "specific proteolytic activity" or "specific activity" of a given preparation is defined as the number of units per mg of protein in the preparation, which is determined by the method described in example 9.

"methylotrophic yeasts" are defined as yeasts that are capable of using methanol as a carbon source. The term also includes laboratory strains thereof. If the methylotrophic yeast strain is auxotrophic and therefore needs to be supplemented with an auxiliary carbon-containing substance such as histidine (in case the methylotrophic yeast strain is not able to synthesize a sufficient amount of this amino acid), the auxiliary substance is considered as a nutrient and not as a carbon source.

A "vector" is defined as DNA that contains (i.e., carries) and retains a DNA fragment of the invention, including, for example, phages and plasmids. These terms are understood by those skilled in the art of genetic engineering. The term "expression cassette" refers to a nucleotide sequence encoding a proprotein operably linked to a promoter and a terminator. For vectors comprising an expression cassette, the terms "vector" and "expression vector" are used as synonyms.

The term "oligonucleotide" is used for nucleic acid molecules, DNA (or RNA) having a length of less than 100 nucleotides.

"transformation" means the introduction of DNA into an organism, i.e.a host organism, so that the DNA is replicable either as an extrachromosomal element or by chromosomal integration.

The term "expression" and the verb "expression" refer to the transcription of a DNA sequence in a host organism and/or the translation of the transcribed mRNA, resulting in a pre-protein, i.e. not including post-translational processing.

A nucleotide sequence "encodes" a peptide or protein when at least a portion of the nucleic acid, or its complement, can be directly translated to provide the amino acid sequence of the peptide or protein, or when the isolated nucleic acid can be used alone or as part of an expression vector to express the peptide or protein in vitro, in a prokaryotic host cell, or in a eukaryotic host cell.

A "promoter" is a regulatory nucleotide sequence that stimulates transcription. These terms are understood by those skilled in the art of genetic engineering. Similar to a promoter, a "promoter element" stimulates transcription but constitutes a sub-fragment of a larger promoter sequence.

The term "operably linked" refers to the association of two or more nucleic acid fragments on a single vector such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (i.e., a nucleotide sequence that encodes a protein or pre-protein) when the promoter is capable of affecting the expression of that coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter.

The term "polypeptide" or "protein" refers to a polymer composed of more than 90 amino acid monomers linked by peptide bonds. The term "peptide" refers to an oligomer consisting of 90 or fewer amino acid monomers linked by peptide bonds. A "peptide bond" is a covalent bond between two amino acids in which the alpha-amino group of one amino acid is bound to the alpha-carboxyl group of another amino acid.

The term "pro-protein", "pro-form", "zymogen", "trypsinogen", "pre-protein" or "pre-pro-protein" refers to the primary translation product which is the precursor of the mature protein, i.e.in this case the protein is obtained from post-translational processing of the pre-protein.

The term "post-translational processing" refers to the modification steps to which a pre-protein or pre-pro-protein is subjected, such that the mature protein is obtained in the cell or extracellular compartment.

A "signal peptide" is a cleavable signal sequence present in an amino acid of a pre-protein or pre-pro-protein form of a secretable protein. Proteins transported (i.e., secreted) through the cell membrane typically have an N-terminal sequence rich in hydrophobic amino acids, typically about 15 to 30 amino acids in length. Sometimes during the process of crossing the membrane, the signal sequence is cleaved by signal peptidases (Alberts, b., Johnson, a., Lewis, j., Raff, m., Roberts, k., Walter, P. (eds.), Molecular Biology of the Cell, 4 th edition, 2002, Garland Science Publishing). Many sources of signal peptides are known to those skilled in the art and may include, for example, the amino acid sequence of the alpha-factor signal peptide from Saccharomyces cerevisiae, and the like. Another example is a polypeptide according to SEQ ID NO: 5 (positions 1-16). In general, the pre-protein N-terminus of essentially any secreted protein is a potential source of a signal peptide suitable for use in the present invention. The signal peptide may also be bipartite, comprising two signal peptides directing the pre-protein to the first and second cellular compartments. The bisecting signal peptide is gradually cleaved off during the secretory pathway. A specific example is therefore the propeptide of the alpha-factor from Saccharomyces cerevisiae (Waters et al, J.biol.chem.263(1988) 6209-14).

The pre-protein with the N-terminal signal peptide is directed into the "secretory pathway". The secretory pathway involves the process of post-translational processing and ultimately leads to protein secretion. Glycosylation and disulfide bond formation are processes that are part of the pre-secretory pathway. In this document, it is understood that proteins secreted by methylotrophic yeast strains have crossed the secretory pathway.

Detailed Description

All trypsin-like serine proteases share substrate preference for the basic residues lysine or arginine. Although the amino acid exchange mutation at the serine corresponding to position 190 of human pancreatic trypsin results in the desired shift in artificial substrate specificity, the result is a decrease in proteolytic activity.

In addition, evaluation of human trypsin mutants showed that the observed shift in specificity for arginine residues using artificial substrates could not be assigned to pre-proinsulin processing. Example 1 illustrates the transformation of pre-proinsulin with a human serine 190 alanine trypsin mutant. During the reaction, a large number of by-products are formed, mainly the B30-des-Thr-and A0-components.

The extent to which this effect applies to all trypsin-like serine proteases has not been shown. One way to address this problem is to introduce a Ser-Ala exchange mutation into the amino acid sequences of other mammalian trypsin species at a position where the respective polypeptide sequence corresponds to position 190 of human pancreatic trypsin isoform I. After doing so, the inventors surprisingly found that such exchange mutations in porcine pancreatic trypsin increase the cleavage site selectivity for arginine in pre-proinsulin processing, and at the same time maintain a higher level of proteolytic activity.

Methods for substituting one or more amino acid residues in a protein are well known to those skilled in the art. Example 2 shows how amino acid exchange mutations can be engineered at the level of the coding DNA sequence. However, other approaches are possible. In the present invention, as in Seq ID NO: 4, the synthetic nucleotide sequence coding for the Ser172Ala mutant of porcine pancreatic trypsin is expressed in a microbial host organism.

Preferably, the trypsin variant is produced as a heterologous protein in a microbial host organism, such as bacteria and fungi. The existence of bacterial expression systems for a variety of prokaryotic hosts such as e.coli (e.coli), Bacillus (Bacillus) and Staphylococcus (Staphylococcus) species, to name a few, is well known to those skilled in the art. Even more preferred microbial host organisms are fungi. An example of a preferred fungal genus is Aspergillus (Aspergillus). But even more preferred are yeast species such as Saccharomyces (Saccharomyces) or Schizosaccharomyces (Saccharomyces). But even more preferred are strains of methylotrophic yeast species.

Methylotrophic yeasts have the biochemical pathway necessary for methanol utilization and are classified into 4 genera based on cell morphology and growth characteristics: hansenula, Pichia, Candida, and Torulopsis; the most highly developed methylotrophic host systems used Pichia pastoris (Komagataella pastoris) and Hansenula polymorpha (Pichia angusta).

Expression of heterologous proteins in yeast is described in US 5,618,676, US 5,854,018, US 5,856,123 and US 5,919,651.

Yeast organisms produce a number of proteins that are synthesized intracellularly but function extracellularly. These extracellular proteins are called secreted proteins. The initially secreted protein is expressed inside the cell in the form of a precursor, pre-protein or pre-pro-protein comprising an N-terminal signal peptide which ensures efficient targeting of the expressed product to the secretory pathway of the cell, across the membrane of the endoplasmic reticulum. The signal peptide is typically cleaved from the desired product during translocation. Cleavage is achieved proteolytically by a signal peptidase. Signal peptidases recognize and cleave specific sub-sequences of signal peptide amino acids. This sub-sequence is called the signal peptidase cleavage site. Once in the secretory pathway, the protein is transported into the golgi apparatus. Proteins are distributed from the golgi apparatus to the plasma membrane, lysosomes and secretory vesicles.

In contrast to intracellular proteins, secreted proteins are subject to different environmental conditions. Part of the secretory pathway process is the stabilization of mature extracellular proteins. Thus, pre-proteins that traverse the secretory pathway of yeast undergo specific post-translational processing. For example, processing may include the generation of disulfide bonds to form intramolecular crosslinks. In addition, certain amino acids of the protein may be glycosylated.

Several methods have been proposed for expressing and secreting proteins in yeast that are heterologous to the yeast. EP 0116201 describes a method for transforming a protein heterologous to yeast with an expression vector having DNA encoding the desired protein, a signal peptide and a peptide acting as a cleavage site for a signal peptidase. A culture of the transformed organism is prepared and cultured, and the protein is recovered from the culture medium. Suitable signal peptides were found as alpha-factor signal peptides from Saccharomyces cerevisiae for use in yeast cells (US 4,870,008).

During secretion, the yeast enzyme KEX-2 is a signal peptidase which recognizes the lysine-arginine sequence as its cleavage site in the pre-protein. KEX-2 cleaves at the junction of the desired protein sequence. Thus, the desired gene product is released and has no leader portion, i.e., the signal peptide of the pre-protein. KEX-2 endoprotease was originally characterized by Saccharomyces yeasts, in which it specifically processes the precursor of the conjugative alpha-factor and the toxin-releasing factor (Julius, D., et al, Cell 37(1984) 1075-1089). Methylotrophic Yeast species such as Pichia pastoris share KEX-2-type proteases with Saccharomyces cerevisiae (similar roles and functions) (Werten, M.W., et al, Yeast 15(1999) 1087-.

A well established methylotrophic yeast species exemplarily described for use as a host for high levels of recombinant proteins is pichia pastoris (US 4,683,293, US 4,808,537, US 4,812,405, US 4,818,700, US 4,837,148, US 4,855,231, US 4,857,467, US 4,879,231, US 4,882,279, US 4,885,242, US 4,895,800, US 4,929,555, US 5,002,876, US 5,004,688, US 5,032,516, US 5,122,465, US 5,135,868, US 5,166,329, WO 00/56903). In the absence of glucose, pichia pastoris uses methanol as a carbon source, which at the same time serves as a marker for methylotrophic organisms. In SEQ ID NO: the Alcohol Oxidase (AOX) promoter given in 7 controls the expression of alcohol oxidase, which catalyzes the first step in methanol metabolism. Typically, 30% of the total soluble protein in methanol-induced cells is alcohol oxidase. Some pichia expression vectors carry the AOX1 promoter and use methanol to induce high level expression of the desired heterologous protein. The expression construct is also integrated into the pichia pastoris genome, resulting in a transformed and genetically stable host.

Methylotrophic yeast strains such as pichia pastoris can be manipulated using an expression vector encoding a heterologous pre-protein (comprising a signal peptide or a signal peptide with a signal peptidase cleavage site, and the desired protein) to secrete the desired product into the growth medium from which the secreted protein can be purified.

It may be advantageous to generate nucleotide sequences encoding pre-proteins with substantially different codon usage. Codons are selected to increase the rate at which pre-protein expression occurs in a particular yeast expression host, depending on the frequency with which particular codons are used by the host. Other reasons for substantially altering the nucleotide sequence encoding the pre-protein without altering the encoded amino acid sequence include the production of RNA transcripts having more desirable properties, such as greater half-life, than transcripts produced from naturally occurring sequences.

Example 3 illustrates the cloning steps to provide an expression vector encoding a trypsin variant. Using a vector comprising a nucleotide sequence encoding a pre-protein capable of expression (e.g., operably linked to a promoter or promoter element and to a terminator or terminator element, and to sequences required for sufficient translation), the host organism is transformed with the vector and transformants are selected.

On the one hand, expression yield depends on the appropriate targeting of the desired product, e.g.to the secretory pathway via a signal peptide, such as the alpha-factor signal peptide from Saccharomyces cerevisiae or the porcine signal peptide of native porcine pancreatic trypsinogen. Example 4 provides a transformed microbial host and example 5 demonstrates how expression of the trypsin variant is achieved. Thus, the primary translation product includes a signal peptide that directs the polypeptide to the secretory pathway. Example 6 illustrates the measurement of trypsin activity in the supernatant of transformed methylotrophic yeast.

On the other hand, expression yield can be increased by increasing the dose of the gene encoding the desired product. Thus, the copy number of the expression construct, which is an expression vector or an expression cassette, is amplified in the host organism. One way to achieve this is by multiple transformation of the expression vector encoding the desired product. Another way is to introduce the gene encoding the desired product into the host organism using a first and a second expression vector, wherein the second expression vector is based on a selection marker different from the one used in the first expression vector. Even when the host organism already carries multiple copies of the first Expression vector, a second Expression vector encoding the same desired product can be introduced (U.S. Pat. No. 5,324,639; Thill, G.P., et al, Positive and Negative Effects of Multi-Copy Integrated Expression in Pichia pastoris, International Symposium on the genetics of microorganisms 2(1990), p.477-490; Vedvick, T.et al, J.Ind.Microbiol.7(1991)197- & 201; Werten, M.W., et al, Yeast 15(1999)1087- & 1096). Example 7 describes how the expression yield of trypsin variants can be increased to provide yeast clones for industrial scale production.

Transformants were analyzed repeatedly for the yield of recombinant protein secreted into the growth medium. The transformants secreting the highest amount of enzymatically active recombinant protein were selected.

Secretion of the porcine trypsin variant into the growth medium directs the mature recombinant protein to the extracytoplasmic space from which it diffuses into the growth medium. Transformed methylotrophic yeast grown in liquid medium secretes the recombinant porcine pancreatic trypsin variant into the liquid growth medium (i.e., liquid medium). This allows a very efficient separation of yeast biomass and recombinant proteins using, for example, filtration techniques. Thus, recombinant porcine trypsin variants purified from this source are very efficiently separated from other enzymatic activities, such as ribonuclease or other (non-trypsin) protease activities.

Thus, a first preferred embodiment of the invention is a variant obtained by amino acid substitution of porcine pancreatic trypsin isoform I, wherein the amino acid serine is substituted with the amino acid residue alanine in position 172 to form a variant of porcine pancreatic trypsin with trypsin activity, said positions being according to SEQ ID NO: 1, numbered beginning at the N-terminus.

Preferably, the variant porcine pancreatic trypsin has increased cleavage site selectivity for hydrolytic cleavage at the carboxyl group of the amino acid arginine, but not for hydrolytic cleavage at the carboxyl group of the amino acid lysine.

More preferably, the isolated variant exhibits increased selectivity when an insulin precursor polypeptide or an analogue thereof is used as a substrate.

In yet another preferred embodiment of the invention, the specific proteolytic activity of the variant porcine pancreatic trypsin is 100% compared to wild-type porcine pancreatic trypsin. Thus, the specific trypsin activity of the recombinant porcine pancreatic trypsin variant is 100% compared to the wild-type form of unaltered porcine pancreatic trypsin when produced and purified under equivalent conditions.

Another preferred embodiment of the present invention is a method of producing a variant of porcine pancreatic trypsin, comprising the steps of (a) providing a vector comprising a nucleotide sequence encoding the variant of porcine pancreatic trypsin, (b) transforming a microbial host strain with the vector, (c) culturing the transformed microbial host strain in a growth medium comprising nutrients, whereby the microbial host strain expresses the variant of recombinant porcine pancreatic trypsin, and (d) purifying the variant of recombinant porcine pancreatic trypsin from the microbial host strain and/or the growth medium.

The translation efficiency of the heterologous protein may be enhanced by adapting the codons of the nucleotide sequence encoding the heterologous protein according to the preferred codons in the host organism. Thus, in a preferred embodiment of the invention, the nucleotide sequence encoding the recombinant porcine pancreatic trypsin variant is SEQ ID NO: 4.

in an even more preferred embodiment of the invention, (a) the vector comprises a nucleotide sequence encoding a pre-protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, (b) the microbial host strain is a methylotrophic yeast strain, (c) the growth medium comprises methanol as a carbon source, (d) the methylotrophic yeast strain expresses and secretes the recombinant porcine pancreatic trypsin variant, and (e) the porcine pancreatic trypsin variant is purified from the growth medium.

Eukaryotic signal peptides of yeast origin and non-yeast origin may be used for the same purpose, except as specifically mentioned. Although the signal peptide may be non-cleavable by a signal peptidase, a signal peptidase-cleaving peptide may be inserted into the pre-protein amino acid sequence located between the amino acid sequence of the signal peptide and the amino acid sequence of the variant recombinant porcine pancreatic trypsin polypeptide. Thus, in another highly preferred embodiment of the invention, the signal peptide comprises a signal peptidase cleavage site adjacent to the first (N-terminal) amino acid of the recombinant porcine pancreatic trypsinogen.

In another preferred embodiment of the invention, the nucleotide sequence encoding the pre-protein is operably linked to a promoter or promoter element. Preferably, the vector is a plasmid capable of replication as an episome in the methylotrophic yeast strain. More preferably, the artificial chromosome capable of replication in the methylotrophic yeast strain comprises a vector. However, it is most preferred that the chromosome of the methylotrophic yeast strain comprises the vector as an integral whole.

Thus, in a preferred method of using methylotrophic yeast strains, and in particular in pichia pastoris strains, the vector encodes the amino acid sequence of a porcine pancreatic trypsinogen pre-protein variant into the secretory pathway.

In a more preferred embodiment of the invention, the methylotrophic yeast strain is a Hansenula, Pichia, Candida or Torulopsis species. Very preferably, the methylotrophic yeast strain is selected from the group consisting of Pichia pastoris, Hansenula polymorpha, Candida boidinii and Torulopsis glabrata. Even more preferably, the methylotrophic yeast strain is a Pichia pastoris strain with American type Culture Collection accession number 76273 or a derivative thereof.

Another preferred embodiment of the present invention is a pichia pastoris strain having a chromosome containing a vector comprising a nucleotide sequence encoding a pre-protein consisting of a recombinant porcine pancreatic trypsin variant and a signal peptide, having the amino acid sequence shown in SEQ ID NO: 7 or a promoter element thereof, is operably linked to the pichia pastoris AOX1 promoter.

The person skilled in the art is aware of the fact that the yield of a secreted heterologous protein (e.g. a variant of porcine pancreatic trypsin) from a growth medium, such as a liquid growth medium, can be increased when the copy number of the nucleotide sequence encoding the pre-protein from which the heterologous protein is expressed and secreted increases. Thus, the yield of secreted heterologous protein from the growth medium can be increased when the vector copy number is increased in the genome of the methylotrophic yeast strain. For example, the Copy number of a vector may be increased by subjecting a methylotrophic yeast strain to repeated transformation of the vector and repeated selection cycles using increasing concentrations of a selection agent against which a selectable marker contained in the vector confers resistance (U.S. Pat. No. 5,324,639; Thill, G.P., et al, Positive and Negative Effects of Multi-Copy Integrated Expression in Pichia pastoris, International Symposium on the genetics of microorganisms 2(1990), p. 477-490; Vedvick, T.T., et al, J.Ind.Microbiol.7(1991) 197-201).

An example of a selectable marker is the Sh ble gene, which is Zeocin^TMResistance genes (Drocoort, D., et al, Nucleic Acids Res.18(1990) 4009; Carmels, T., et al, curr. Genet.20(1991) 309-. The protein encoded by the Sh ble gene binds stoichiometrically to Zeocin^TMAnd has strong affinity. Zeocin^TMInhibits its toxic activity, thereby selecting transformants containing the Sh ble gene. It is known to the person skilled in the art to add Zeocin as a selection agent in the culture medium^TMThe concentration of (a) is selected for an increase in the copy number of the vector expressing the Sh ble gene. Thus, it is advantageous to use a vector using the Sh ble gene as a selection marker to generate a multiple transformant of a methylotrophic yeast strain containing multiple copies of the vector by repeated transformation. More advantageously, the transformation is repeated and the selection of more resistant transformants is repeated until no more Zeocin is available for the transformed methylotrophic yeast strain^TMFurther increase in resistance to aqueous, or no increase in Zeocin in selection Medium is possible anymore^TMAnd (4) concentration.

Those skilled in the art are familiar with the purification by chromatography of recombinantly expressed and secreted porcine pancreatic trypsin (Funakoshi, A., et al, J.Biochem. (Tokyo)88(1980) 1113-H1138; Paudel, H.K., and Liao, T.H., J.biol chem.261(1986) 16006-16011; Nefsky, B., and Bretscher, A., Eur.J.Biochem.179(1989) 215-219). Preferably, the porcine pancreatic trypsinogen variant in the growth medium is purified using ion exchange chromatography. The downstream processing steps to produce the purified product are described in example 8. Wild-type porcine pancreatic trypsin isoform I was similarly generated, except that the wild-type coding sequence was used to construct the expression vector.

Another preferred embodiment of the invention is a variant of porcine pancreatic trypsin isoform I by one of the methods described above. Another embodiment of the invention is the use of a variant of porcine pancreatic trypsin for pre-proinsulin processing. The benefits of using the Ser172Ala variant of porcine trypsin for enzymatic cleavage of different pre-proinsulin are described in examples 10-12. The following examples, references, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that changes may be made in the methods described without departing from the spirit of the invention.

Description of the drawings

FIG. 1: dot plots of major trypsin cleavage of pre-pro-insulins of human insulin, insulin glargine and insulin glulisine. Filled triangles indicate cleavage sites that produce product, open triangles indicate cleavage sites that produce by-products. The disulfide bond of pre-pro insulin is not shown.

FIG. 2: pre-proinsulin cleavage with recombinant, wild-type human trypsin (example 1).

FIG. 3: cleavage with recombinant, serine 190 alanine human trypsin variant pre-proinsulin (example 1).

FIG. 4: map of plasmid pTry-Ser172Ala, which is a derivative of the commercially available plasmid pPICZ. alpha.A (Invitrogen), which is endowed with resistance to Zeocin^TMResistance of (2). The insert indicating TrySer172Ala is a synthetic DNA sequence encoding a recombinant porcine secreted trypsinogen variant carryingSer172Ala amino acid substitution, and is fused to a nucleotide sequence encoding an alpha-factor signal peptide from Saccharomyces cerevisiae. "AOX 1-Prom" refers to the Pichia pastoris AOX1 promoter, and "Term" refers to the Pichia pastoris AOX1 terminator.

FIG. 5: cleavage of pre-proinsulin (human insulin) with native, recombinant porcine trypsin is shown (example 10, table 1).

FIG. 6: cleavage with S172A variant porcine trypsin pre-proinsulin (human insulin) is shown.

In general, in the following examples, the methods proposed and described in the operating manual "Pichia expression Kit" version M01110225-0043, "pPICZ A, B and C" versions D11080125-0148, "pPICZ α A, B and C" versions E01030225-0150 and "pPIC 9K" version E03040225-0106, from Invitrogen (Invitrogen) were applied. Reference is also made to other vectors, yeast strains and media mentioned therein. The basic method of Molecular biology is used as described in Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3 rd edition, CSHL Press, 2001.

HPLC method:

stationary phase: nucleosil 120-5C18, Macherey & Nagel, 250X 4 mm; mobile phase A: 45mM sodium phosphate buffer (pH2, 5), 315mM NaCl, 25% (v/v) acetonitrile; mobile phase B: 45mM sodium phosphate buffer (pH2, 5), 55mM NaCl, 65% (v/v) acetonitrile; gradient: linear, from 6% B phase to 10% B phase in 30 minutes.

The following examples are intended to illustrate the invention, but not to limit it.

Example 1: cleavage of pre-proinsulin by recombinant human wild-type trypsin and serine 190 alanine human trypsin variant

These experiments were performed at 8 ℃ and pH 8.3 (buffered solution) and were performed at maximum on a 50mL scale.

The PPI solution is charged into a suitable isothermal reaction vessel and the reaction is started by adding the enzyme preparation. Sampling samples after a certain time interval; the enzymatic reaction was immediately stopped by acidifying the sample solution by 1N or 2N HCl solution. The concentration of each product was determined by HPLC.

Example 2: mutagenesis of synthetic nucleotide sequences encoding porcine pancreatic trypsinogen

Generally, standard methods of Molecular biology as described in Sambrook, Fritsch & Maniatis, Molecular Cloning, laboratory Manual, 3 rd edition, CSHL Press, 2001 are applied. The method described below is a specific application of a very conventional method known as "site-directed mutagenesis".

The mutations were generated in a site-directed fashion using the polymerase chain reaction. To mutate the desired codon (i.e., base triplet), a pair of complementary single-stranded DNA oligonucleotides representing the variant portion of the synthetic nucleotide sequence encoding porcine pancreatic trypsinogen were designed and synthesized. In addition to the triplet sequence to be mutated, the single-stranded DNA oligonucleotide hybridizes with the nucleotide sequence set forth in SEQ ID NO: 2 are identical or complementary. Typically, DNA oligonucleotides have a length of about 20 to 45 nucleotides; the triplet sequence to be mutated or its complement is located in the middle portion of the DNA oligonucleotide comprising it and is flanked on both sides by about 10 to 12 nucleotides. The DNA oligonucleotide is designed such that it hybridizes to "wild-type" recombinant porcine pancreatic trypsinogen DNA (according to SEQ ID NO: 2), resulting in a hybrid molecule with a central mismatch but complete base pairing on both sides of the mismatch, including the 5 'and 3' ends of each DNA oligonucleotide.

In addition, two single-stranded DNA oligonucleotide primers are provided, wherein the first (referred to as "5' trypsin") (SEQ ID NO: 8) comprises the nucleotide sequence of SEQ ID NO: 2, and the second (termed "3 trypsin") (SEQ ID NO: 9) comprises a sequence identical to the sequence of SEQ ID NO: 2, and 12 nucleotides at the 3' -end. Both primers were designed to contain restriction enzyme cleavage sites. Thus, the first and second primers are extended and comprise sequences identical to SEQ ID NO: 2, flanking the synthetic nucleotide sequence. "5 'trypsin" contains EcoRI and 3' Xba I sites.

Nucleotide sequences encoding variants obtained by amino acid substitutions of the wild-type mature porcine pancreatic trypsin protein were synthesized by several PCR-based steps.

Performing the first and second PCR using as a template a double stranded DNA comprising a sequence according to SEQ ID NO: 2, which is present as an insert in a vector. The vector sequences flanking the insert were those that annealed during PCR so that the primers "5 'trypsin" and "3' trypsin" matched perfectly. The first PCR was performed using a pair of primers consisting of a "5' trypsin" primer and a first single-stranded DNA oligonucleotide comprising a mutated (i.e., variant triplet) sequence, so that both primers anneal to opposite template DNA strands. Thus a second PCR was performed using the "3' trypsin" primer and a second single-stranded DNA oligonucleotide complementary to the first. Thus the first and second PCR yielded two direct products: 5 'and 3' portions of the nucleotide sequence encoding the recombinant variant of porcine pancreatic trypsin, wherein the 5 'portion carries a mutated sequence at its 3' end and vice versa and the 3 'portion carries a mutated sequence at its 5' end.

The two resulting intermediate amplification products were analyzed by agarose Gel electrophoresis, the desired fragments were sheared and the DNA was isolated from the agarose block using the "QIAquick Gel Extraction Kit" Qiagen, cat No. 28704.

A third PCR was then performed to fuse the two portions. To this end, the two parts were combined in a single PCR and 5 PCR cycles were performed without adding any additional upstream and downstream primers. In these cycles, some full-length products were formed, with the annealing temperatures used being calculated for the overlapping sequences of the 5 'and 3' portions. Subsequently, primers "5 'trypsin" and "3' trypsin" were added and an additional 25 PCR cycles were performed, wherein the annealing temperature used here corresponds to the added primer with the lower melting temperature.

The mutated full-length DNA fragment was subsequently inserted using the "PCR cloning kit-blunt-ended" (Roche Diagnostics GmbH, Mannheim; cat # 1939645). The DNA fragments were verified by restriction enzyme analysis and sequencing. The validated DNA fragments were then excised by cleavage with XhoI and NotI and inserted into pichia pastoris expression vectors, which were cleaved with the same restriction enzymes (see example 3 and example 5).

Ser172 Ala: in SEQ ID NO: the base triplet "TCT" found at position 528-531 of 2 was replaced by "GCT". For this purpose, the DNA oligonucleotide "5 '-Try-Ser 172 Ala" (SEQ ID NO: 10) was used as a primer in combination with "3' -trypsin" in the first PCR, and "3 '-Try-Ser 172 Ala" (SEQ ID NO: 11) was used as a primer in combination with "5' -trypsin" in the second PCR. The isolated intermediate fragments were then used for a third PCR, resulting in full-length products.

Example 3: cloning of an artificial DNA encoding a variant recombinant porcine pancreatic trypsinogen in a pPICZ alpha A-derived expression vector

The DNA fragment encoding the variant recombinant porcine pancreatic trypsinogen generated from the PCR fragment was cut with EcoRI and XbaII (Roche diagnostics GmbH) (see example 2). The fragments were isolated using the "QIAauick gel extraction kit" according to the manufacturer's instructions.

The fragment was ligated into the pPICZ α A vector, fusing the nucleotide sequence encoding the variant recombinant porcine pancreatic trypsinogen to the nucleotide sequence encoding the α -factor signal peptide from Saccharomyces cerevisiae. The vector was similarly cleaved with EcoRI and XbaI and isolated prior to ligation.

The subsequent cloning step inserts the nucleotide sequence encoding the variant recombinant porcine pancreatic trypsinogen directly and in-frame after the nucleotide sequence encoding the s.cerevisiae alpha-factor signal peptide.

As set forth in SEQ ID NO: 6 is under the control of the Pichia pastoris AOX-1 promoter (SEQ ID NO: 7), which is inducible by methanol, for example in Pichia pastoris.

Construction was carried out according to the manufacturer's instructions by ligating 20ng of the linearized vector fragment (volume 1. mu.l), 100ng of the cleaved PCR fragment (volume 3. mu.l) in a total volume of 10. mu.l and in the presence of T4DNA ligase (Roche diagnostics, Inc.) at 16 ℃. Subsequently 5. mu.l of the ligation preparation were used to transform competent E.coli XL1Blue cells (Stratagene) in a total volume of 205. mu.l. After incubation on ice for 30 minutes, the cells were heat shocked at 42 ℃ for 90 seconds. Subsequently, the cells were transferred to 1ml of LB medium and incubated at 37 ℃ for 1 hour to allow expression of the selectable marker. Aliquots were then plated on LB plates containing 100. mu.g/ml Zeocin and incubated for 15 hours at 37 ℃. Resistant clones were picked, plasmids were isolated (Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3 rd edition, CSHL Press, 2001) and tested by restriction analysis as well as sequence analysis. Construct clones verified as error free and clone artifacts were selected. The expression vector with the variant recombinant porcine pancreatic trypsin and the alpha-factor signal peptide of Saccharomyces cerevisiae was named pTry-Ser172 Ala.

Example 4: transformation of Pichia pastoris with pPICZ alpha A-derived pTry-Ser172Ala expression vector

The host strains used were Pichia pastoris X-33, GS115, KM71H and SMD1168 (Invitrogen). Preferred strains are X-33 and KM 71H. The transformation is aimed at the stable integration of the expression construct into the genome of the host organismIn (1). Initially, 5ml YPD medium (YPD ═ yeast peptone glucose; Invitrogen) was inoculated with a Pichia pastoris colony and pre-cultured overnight at 30 ℃ on a shaker. To prepare transformation competent cells, 100. mu.l of the preculture was added as inoculum to 200ml of fresh YPD medium and grown until OD_600nmUp to between 1.3 and 1.5. The cells were centrifuged at 1500 Xg for 5 minutes and resuspended in 200ml ice-cold (0 ℃) sterile water. The cells were centrifuged again at 1500 × g for 5 minutes and resuspended in 100ml of ice-cold sterile water. The cells were centrifuged once more at 1500 × g for 5 minutes and resuspended in 10ml ice-chilled 1M sorbitol (ICN). Cells prepared in this manner were kept on ice and used immediately for transformation.

The pPICZ α a-derived pTrySer172Ala expression vector to be used for transformation was linearized using SacI restriction endonuclease (roche diagnostics ltd.), precipitated and resuspended in water. Transformation was carried out by using "Gene Pulser II^TN"(BioRad) was performed. For transformation preparation, 80 μ l of pichia pastoris in 1M sorbitol solution was gently mixed with 1 μ g of linearized expression vector DNA and transferred to ice-precooled cuvettes, which were then kept on ice for 5 minutes. Subsequently, the cuvette was transferred to a gene pulser (GenePulser). The electroporation parameters were 1kV, 1 kOmega and 25. mu.G. After electroporation, 1ml of 1M sorbitol solution was added to the cell suspension, which was subsequently inoculated into YPDS plates (YPDS ═ yeast peptone glucose sorbitol, Invitrogen) containing 100 μ g/ml zeocin^TM(Invitrogen) wherein 100-150. mu.l of cell suspension was plated on a single plate. YPDS plates were incubated at 30 ℃ for 2-4 days. Yeast clones were transferred to gridded minimal glucose plates. Colonies of these plates were picked and resuspended in sterile water separately. The cells were digested with 17.5 units of cytolytic enzyme (Roche diagnostics GmbH) at 30 ℃ for 1 hour, and then frozen at-80 ℃ for 10 minutes. The presence of the expression cassettes of the respective pPICZ α a-derived pTrySer172Ala expression vectors was verified by PCR. The term "expression cassette" isRefers to a nucleotide sequence encoding a variant recombinant porcine pancreatic trypsin pre-protein operably linked to the AOX1 promoter and AOX1 terminator, wherein the expression cassettes are from the respective pPICZ α a-vectors used for transformation. For vectors comprising an expression cassette, the terms "vector" and "expression cassette" are synonymous.

Positive clones, i.e. clones verified as positive for the presence of the complete expression cassette stably integrated into the genome, were used for further characterization of variant recombinant porcine pancreatic trypsin expression.

In addition, a control transformation was performed using the original pPICZ α a vector together with the recipient pichia pastoris KM71H strain. Positive clones were obtained and verified in a similar manner.

Example 5: expression and secretion of variant recombinant porcine pancreatic trypsinogen

A set of positive clones (20-30) transformed with pPICZ α A-pTrySer172Ala expression vector was cultured overnight as shaking cultures in 3ml of BMGY medium (BMGY ═ buffered glycerol complex medium; Invitrogen). The OD of the cultures was then determined before they were passaged in shake flasks₆₀₀Values, each shake flask contained 10ml BMMY medium (invitrogen) at pH3. Preculture was used as inoculum, so that each OD was_600nmIs 1. The cultures were kept on a shaker at 30 ℃ for 30 minutes. In parallel, positive control clones were cultured under the same conditions.

BMMY (BMMY ═ buffered methanol complex medium) medium contains methanol (Mallinckrodt Baker b.v.), an inducer of the AOX-1 promoter that controls transcription of the nucleotide sequence encoding the variant recombinant porcine pancreatic trypsinogen.

Throughout the period of 72 hours, 500. mu.l samples were taken from the flasks at 24-hour intervals. When removing sample aliquots, the culture was also added to 0.5% methanol. Samples of the supernatant growth medium were tested for trypsin enzymatic activity.

Example 6: analysis of variant porcine pancreatic trypsin expression

The OD of the sample aliquot obtained as described in example 5 was first determined_600nm. The cells were then pelleted by centrifugation and the supernatant was stored. Trypsin activity was measured in undiluted supernatants as well as in 1: 10 diluted supernatants.

Although the control clone transformed with the pPICZ α a vector did not produce any measurable trypsin activity in the medium, the pichia strain transformed with the pPICZ α a-pTrySer172Ala expression vector exhibited trypsin activity due to the respective variant of recombinant porcine pancreatic trypsin secreted into the growth medium (i.e., the culture medium). It can thus be concluded that in this case the expression of a recombinant pre-protein comprising the alpha-factor signal peptide of s.cerevisiae enables the secretion of the active enzyme with proteolytic activity.

Example 7: zeocin by multiple transformations and additions^TMConcentration increase expression yield

Repeated electroporation was performed using the same expression vectors as before for yeast clones transformed with pPICZ α A-and pPICZA-derived pDNM expression vectors and found to produce the highest trypsin activity in the supernatant medium. Conditions for electroporation are described in example 4, except that YPDS plates contain increasing concentrations (between 1000 and 2000. mu.g/ml) of Zeocin^TM. The concentration of the antibiotic is increased to select transformants which have integrated into their genomes multiple copies of the respective expression vector. Yeast clones with increased resistance to antibiotics were transferred to gridded minimal glucose plates. Precultures were produced from individual yeast clones as already described in example 5, and expression was measured by determining the trypsin enzymatic activity secreted into the growth medium as described in example 6. Find singleCloning resulted in increased amounts of trypsin activity. Generally, trypsin activity measured in supernatants of pichia strains repeatedly transformed with the respective pPICZ α a-pTry-Ser172Ala expression vectors was 2 to 3 times higher compared to the respective precursor strains which had been transformed only a single time.

Example 8: purification of variants of porcine pancreatic trypsinogen from liquid culture supernatants and activation to form Trypsin variants

The whole fermentation broth was diluted with a buffer solution of ammonium acetate (5-20mM) containing 5-30mM calcium chloride, pH3.5, in a ratio of about 1: 2 to 1: 4. By expanded bed chromatography (McCornick (1993); EP 0699687) using a cation exchanger (e.g.streamline)SP, XL) purification of the trypsinogen variant. Chromatography was performed without prior isolation of yeast cells. Using a packed bed column (bed column) (e.g. SP-Sepharose)XL, ff) for further purification. By adding 20mM CaCl₂In the presence of (c), the autocatalytic activation is started by re-buffering the pH to 7-8. Activation was terminated by changing the pH back to the range of 2-4. The purified trypsin was stored at pH1.5-3 to avoid autoproteolysis.

Example 9: assay for determining specific trypsin activity of purified variant recombinant porcine pancreatic trypsin

Chromozym TRY (Roche diagnostics GmbH) was used at 100mM Tris pH8.0, 20mM CaCl₂The trypsin activity was measured at 25 ℃. Photometric measurements were performed at 405 nm. To distinguish the substrate specificity of arginine from lysine, Chromozym TH (including arginine/Roche diagnostics, Inc.) and Chromozym PL (including lysine/Roche diagnostics, Inc.) were used.

Example 10: cleavage of pre-human proinsulin

All experiments were performed at 8 ℃ and pH 8.3 (buffer or controlled by NaOH dosage) and were performed on a 20mL to 3.5L scale. In some experiments, pre-pro-insulin was also cleaved using native, recombinant porcine trypsin, thereby directly comparing the two enzymes.

The PPI solution is charged into a suitable thermally stable reaction vessel and the reaction is started by adding the enzyme preparation. Sampling samples after a certain time interval; the enzymatic reaction was immediately stopped by acidifying the sample solution by 1N or 2N HCl solution. The concentration of the respective product was determined by HPLC.

Table 1 depicts exemplary results of an experiment

Scale of

Trypsin

Sigma pre-Arg-Ins [ area%]

Sigma Arg-Ins [ area%]

des-Thr [ area%]

Sigma A0 Compound [ area%]

Yield (external standard) [% ]]

3.5L

Natural S172A

1.40.9

68.882.5

7.23.3

10.74.1

84.296.5

Table 1: results of pre-insulin (human insulin) cleavage reactions using native recombinant porcine trypsin and the S172A trypsin variant (using equal amounts of U/gPPI).

As can be seen from Table 1, the use of the S172A trypsin variant reduces the formation of the unwanted by-products des-Thr and A0-compounds and thus increases the amount of valuable Arg-insulin (Arg-Ins) and the respective cleavage yield.

FIG. 5 shows the conversion of pre-pro-insulin with native recombinant porcine trypsin and FIG. 6 shows the conversion of pre-pro-insulin (PPI) with the S172A variant.

Example 11: cleavage of Pre-Pro-insulin

The experimental conditions of example 10 were used. Pre-pro-insulin was also cleaved using native, recombinant porcine trypsin, thereby directly comparing the two enzymes.

HPLC method: as described.

Table 2 describes exemplary results of the experiments performed. The values given mark the points of maximum product formation.

Scale of

Trypsin

Pre-insulin glargine [ area%]

Insulin glargine [ area%]

des-Thr [ area%]

Sigma A0 Compound [ area%]

Yield (external standard) [% ]]

1L

Natural S172A

0.50.4

51.155.5

5.62.6

10.95.1

58.162.9

Table 2: results of pre-insulin (insulin glargine) cleavage reaction using native, recombinant trypsin and S172A variant (using 200U/g PPI).

As can be seen from table 2, the use of the S172A trypsin variant reduced the formation of the unwanted by-products des-Thr and a 0-compounds and thus increased the amount of insulin glargine.

Example 12: cleavage of pre-glutelin

The experimental conditions of example 10 were used. Pre-pro-insulin was also cleaved using native, recombinant porcine trypsin, thereby directly comparing the two enzymes. Table 3 describes exemplary results of the experiments performed. The values given mark the points of maximum product formation.

Scale of	Trypsin	Amount of trypsin [ U/g PPI]	Arg-Glulysine insulin [ area%]	A0-Arg-Glulisine [ area%]	Yield (external standard) [% ]]
						1L	Natural S172A	250250	35.839.2	4.71.2	91.6100.0

Table 3: results of pre-insulin (insulin glulisine) cleavage reaction using native, recombinant trypsin and the S172A trypsin variant.

As can be seen from table 3, the use of the S172A trypsin variant reduced the formation of the unwanted a 0-compound and thus increased the amount of insulin glargine.

Claims (17)

1. A process for preparing insulin, an insulin analogue or an insulin derivative, wherein:

(a) using a nucleic acid sequence consisting of the sequence SEQ id no: 3 the Ser172Ala porcine trypsin cleaves pre-pro-insulin, pre-pro-insulin analogue or pre-pro-insulin derivative,

(b) the resulting cleavage product is isolated and

(aa) if one of the cleavage products obtained is an insulin analogue or an insulin derivative, obtaining the insulin analogue or insulin derivative, or

(bb) those cleavage products which are precursors of insulin, insulin analogs or insulin derivatives are further processed and the insulin, insulin analogs or insulin derivatives obtained from this further processing are isolated and obtained.

2. The method according to claim 1, wherein the insulin is human insulin.

3. The method according to claim 1, wherein the insulin analogue is selected from the group comprising Lys^B28Pro^B29Human insulin, B28Asp human insulin, human insulin wherein the proline at position B28 has been substituted by Asp, Lys, Leu, Val or Ala and Lys at position B29 may be substituted by Pro; AlaB26 human insulin; de-B28-B30 human insulin; de-B27 human insulin and de-B30 human insulin.

4. The method according to claim 1, wherein the insulin analogue is insulin glulisine.

5. The method according to claim 1, wherein the insulin analogue is insulin glargine.

6. The method according to claim 1, wherein said insulin derivative is selected from the group consisting of B29-N-myristoyl-des-B30 human insulin, B29-N-palmitoyl-des-B30 human insulin, B29-N-myristoyl human insulin, B29-N-palmitoyl human insulin, B28-N-myristoyl Lys^B28Pro^B29Human insulin, B28-N-palmitoyl-Lys^B28Pro^B29Human insulin, B30-N-myristoyl-Thr^B29Lys^B30Human insulin, B30-N-palmitoyl-Thr^B29Lys^B30Human insulin, B29-N- (N-palmitoyl-Y-glutamyl) -des-B30 human insulin, B29-N- (N-lithocholyl-Y-glutamyl) -des-B30 human insulin, B29-N- (omega-carboxyheptadecanoyl) -des-B30 human insulin and B29-N- (omega-carboxyheptadecanoyl) human insulin.

7. The process according to any one of claims 1 to 4 and 6, wherein the further processing of those cleavage products which are precursors of insulin, insulin analogues or insulin derivatives comprises cleavage of said products with carboxypeptidase B.

8. The method according to any one of claims 1 to 7, wherein the cleavage with Ser172Ala porcine trypsin is performed at a pH value in the range of 7.5 to 9.5, at a temperature of 1 ℃ to 30 ℃; and terminating the enzymatic reaction by acidifying the sample.

9. The method according to claim 8, wherein the cleavage is performed at a pH of 8.3, a temperature between 8 and 12 ℃, and acidification is performed by adding 1N or 2N HCl solution.

10. Consists of the sequence SEQ id no: 3 Ser172Ala porcine trypsin.

11. Encoding a polypeptide consisting of the sequence SEQ id no: 3, Ser172Ala porcine trypsin.

12. DNA according to claim 11, which is encoded by the sequence SEQ id no: and 4, characterizing.

13. By the sequence SEQ id no: 6 DNA encoding Ser196Ala porcine trypsinogen.

14. A vector comprising the DNA according to any one of claims 11, 12 or 13.

15. Generating a polypeptide consisting of the sequence SEQ id no: 3 a method of Ser172Ala porcine trypsin characterized comprising the steps of:

(a) providing a carrier according to claim 14, wherein said carrier is a non-woven material,

(b) transforming a microbial host strain with the vector,

(c) cultivating the transformed microbial host strain in a growth medium comprising nutrients, whereby the microbial host strain expresses Ser172Ala porcine trypsin or Ser196Ala porcine trypsinogen,

(d) if the expression product of (c) is Ser196Ala porcine trypsinogen, it is converted into mature Ser172Ala porcine trypsin, and

(e) the Ser172Ala porcine trypsin was purified from the microbial host strain and/or the growth medium.

16. The method according to claim 15, wherein the microbial host strain is a methylotrophic yeast strain selected from the group comprising Hansenula, Pichia, Candida and Torulopsis species.

17. The method according to claim 16, wherein the microbial host strain is selected from the group comprising pichia pastoris, hansenula polymorpha, candida boidinii and torulopsis glabrata.