WO2013149729A2 - Proinsulin with enhanced helper sequence - Google Patents

Abstract

The invention provides a polypeptide comprising an insulin precursor and the N-terminal helper sequence MAKR. The helper sequence enhances the expression of the insulin precursor in a recombinant organism and increases the yields of insulin after purification and processing of the insulin precursor. The invention is thus further concerned with a method of producing insulin, in which proinsulin is expressed as recombinant polypeptide in an organism such as E. coli, isolated from the organism and then processed to yield active insulin.

Description

Proinsulin with enhanced helper sequence

Field of the invention: The invention is concerned with a polypeptide comprising an insulin precursor and the N- terminal helper sequence MAKR. The helper sequence enhances the expression of the insulin precursor in a recombinant organism and increases the yields of insulin after purification and processing of the insulin precursor. In addition, the amount of unwanted byproducts is significantly reduced. The invention is thus further concerned with a method of producing insulin, in which proinsulin is expressed as recombinant polypeptide in an organism such as E. coli, isolated from the organism and then processed to yield active insulin.

General remarks:

Insulin is a peptide hormone and regulates the blood glucose level. Therefore, insulin is administered to patients suffering from diabetes mellitus, a metabolic disorder characterized by an inadequate supply of insulin. According to the World Health Organization (WHO) some 320 million people will suffer from diabetes mellitus in 2012. Insulin thera- py is essential to the survival of those with type 1 diabetes and is used to control the symptoms for those patients suffering from type 2 diabetes.

Human insulin consists of two separate chains, which are the A-chain of a length of 21 amino acids and the B-chain of a length of 30 amino acids. The A- and B-chains are joined together by a characteristic pattern of disulfide bridges. In the human pancreas, insulin is produced as a single pre-proinsulin chain in which the prospective A- and B-chains are linked together by the C-peptide and which further contains an N-terminal signal sequence. The formation of native insulin from proinsulin involves folding and the formation of correct disulfide bridges. Proinsulin is then processed proteolytically, which results in the cleavage of the C-peptide and release of the active hormone. Traditionally, insulin was produced from animal sources such as bovine and porcine pancreatic preparations. Insulin produced from animal sources, however, differs from human insulin and thus may elicit an adverse immune reaction. Due to the enormous demand for insulin as medicament, biosynthetic human insulin is manufactured for widespread clinical use using recombinant DNA technology.

Due to high rate of synthesis and rapid growth, the primary source for the manufacturing of biosynthetic recombinant insulin is its production in Escherichia coli (E. coli). There are two major approaches for the generation of recombinant human insulin from E. coli. In one approach, the A- and B-chains of human insulin are expressed separately, converted to their stable S-sulfonate derivatives, and subsequently combined to generate native insulin. The other approach is to produce proinsulin as a recombinant gene product, which is subsequently isolated from the host cell and processed in vitro by proteases to release the C- peptide and yield native insulin. The second approach is preferred, largely as a conse- quence of the requirement for only a single fermentation and subsequent purification protocol, thus it is more efficient than the two-chain combination approach.

However, manufacturing of recombinant proinsulin in E. coli is also accompanied with by several disadvantages. Upon expression in host cells high molecular weight aggregates are formed, often referred to as "inclusion bodies", which result from the inability of the expressed proteins to fold correctly in an unnatural cellular environment. The protein is present in the insoluble inclusion bodies in denatured form, thus requiring the use of detergents and denaturants to isolate and solubilize the protein. The isolated protein must subsequently be refolded in vitro. This includes the formation of the correct disulfide bridges and processing by proteases to convert the correctly folded proinsulin precursor into insulin.

Furthermore heterologous expression in host cells often leads to unspecific degradation of the heterologous protein, resulting in low protein yield and multiple degradation products. The cumbersome and laborious procedures for the isolation of recombinant human insulin from E. coli involving proinsulin expression, isolation, refolding and processing often results in low yields of active product. Expression yields often depend on the stability of the induced protein as it is affected by the protein sequence. A major factor influencing the half-life of a protein is the N- terminus, as described by the N-end rule (Tobias et al. (1991), Science, 254(5036), 1374- 7). The residues arginine, lysine, leucine, phenylalanine, tyrosine and tryptophan at the amino terminus (N-terminus) tend to decrease the half-life of the protein, i.e., the half-life can be in the order of two minutes, whereas other residues provide proteins having a half- life of more than ten hours for a protein differing only in the N-terminal amino acids.

Another approach to form more stable recombinant protein is to fuse the protein to another protein which is naturally present or which is easily expressed in the host organism. EP 0 871 474 Bl describes a method of insulin production in E. coli, in which the proinsulin polypeptide is fused to a superoxide dismutase (SOD) as leader sequence. After partial purification, the hybrid polypeptide is folded and subsequently processed with trypsin and carboxypeptidase to cleave off the leader peptide and the C-chain concomitantly. Methods of isolating insoluble recombinant protein from inclusion bodies are well known in the prior art. These methods however, mostly result in low yield of active protein. Accordingly, a need exists to increase the yield of active protein such as human proinsulin after expression, isolation and processing of the recombinant protein from microorganisms such as E. coli.

Although it was known from the N-end rule in bacteria that some N-terminal amino acids may considerably increase the half-life time of proteins compared to other amino acids, it has recently been found out by the inventors of the present invention that the amino acid sequence MAK located at the N-terminus of a polypeptide comprising an insulin precursor significantly increases the final cell density of a culture of a heterologous organism expressing the recombinant polypeptide. Additionally, it has been found out that the amino acid sequence MAK located at the N-terminus of a polypeptide comprising an insulin precursor also increases expression and stability of the recombinant polypeptide.

However, in spite of the significant improvements achieved by the N-terminal MAK helper sequence, it has been noticed by the inventors of the present invention that processing the insulin precursor including the MAK helper sequence to the final insulin product still suffers from the generation of unwanted by-products, such as des30insulin, in which the insulin chain B is cleaved after lysine 29, thus removing the amino acid threonine at position 30. The generation of by-products, however, lead to a reduction of final insulin product after processing. Further, the by-products have to be separated and removed from the insulin product, which often is a cost and time consuming process with high demands on purification effectiveness. Therefore, there remained a need for increasing the yield of active protein such as human proinsulin after expression, isolation and processing of the recombinant protein, i.e. to increase the amount of insulin product after processing. In addition, it has been observed that enzymatic cleavage of the MAK helper sequence from the insulin chain was not always satisfactory.

Therefore, although expression of an insulin precursor could be greatly enhanced by the N- terminal MAK helper sequence, there still remained a need for improvement of the method for producing insulin.

Object and summary of the invention:

The underlying technical problem of the present invention thus is to achieve the same im- provements achieved by the MAK helper sequence, i.e. enhance the expression of a proinsulin precursor in a heterologous organism and thereby increasing the yield of active recombinant insulin obtained from the proinsulin after expression, but to reduce the amounts of unwanted by-products. In particular, it is the object of the present invention to provide a recombinant polypeptide comprising the sequence of proinsulin, which is expressed in E. coli in high amounts and which can thus be easily isolated from inclusion bodies and which can further be easily processed into active insulin. Thus, it was another object of the present inventors to develop a process for obtaining recombinant active insulin, preferably active human insulin in high amounts. In addition, the amount of unwanted by-products should be reduced. The solution of the underlying technical problems is surprisingly that the yield of recombinant proinsulin can be increased by fusing to the amino terminus (N-terminus) of the amino acid chain of the proinsulin precursor a helper sequence which consists of the amino acid sequence methionine, alanine, lysine and arginine, which is written in the standard amino acid one-letter code as MAKR.

It has surprisingly been found out by the inventors of the present invention that the amino acid sequence MAKR located at the N-terminus of a polypeptide comprising an insulin precursor significantly increases the final cell density of a culture of a heterologous organism expressing the recombinant polypeptide. Additionally, it has been found out that the amino acid sequence MAKR located at the N-terminus of a polypeptide comprising an insulin precursor also increases expression and stability of the recombinant polypeptide. Further, compared to the MAK helper sequence developed recently, the improved helper sequence MAKR further reduces the presence of unwanted by-products, thereby increasing the amount of active recombinant polypeptide after processing.

The findings of the present invention are particularly surprising since it could be expected that the single arginine residue of the MAKR sequence could be left attached to the insulin after enzymatic processing. However, this initial fear turned out to be groundless. In contrast, enzymatic cleavage of the MAKR helper sequence turned out to be much more effi- cient than cleavage of the MAK helper sequence.

The present invention thus provides a polypeptide comprising,

i. a first peptidyl fragment consisting of the N-terminal amino acid

methionine, alanine, lysine and arginine (MAKR), and

ii. a second peptidyl fragment, which is an insulin precursor. In a preferred embodiment, the insulin precursor comprises insulin chains A and B, and most preferably is human proinsulin.

The invention further provides an isolated nucleic acid, which preferably is DNA, and comprising a nucleotide sequence encoding the polypeptide comprising the N-terminal MAKR sequence and the insulin precursor.

The present invention further provides a recombinant cell such as a recombinant E. coli cell, which contains the nucleic acid encoding an amino acid sequence comprising the N- terminal MAKR sequence and the insulin precursor.

The invention is further directed to a method of producing a polypeptide, the method comprising growing a recombinant cell such as an E. coli cell containing a nucleic acid such that the polypeptide encoded by the nucleic acid is expressed by the cell, and recovering the expressed polypeptide from the cell.

In a preferred embodiment, the method of producing insulin comprises the steps of

a. expressing the polypeptide comprising the N-terminal MAKR sequence and the insulin precursor in Escherichia coli,

b. isolating the polypeptide from the recombinant cell,

c. subjecting the isolated polypeptide to a folding process which permits correct folding of the insulin precursor, and

d. subjecting the polypeptide to enzymatic processing to yield active insulin. Brief description of the drawings:

Figure 1 shows a MALDI-TOF analysis for processed and purified MAKR-insulin precursor according to the present invention. Figure 2 shows the MALDI-TOF analysis of processed and purified MAK-insulin precursor according to a comparison example. Detailed description of the invention:

In the present invention the term "heterologous expression" means that the protein is experimentally put into a cell that does not normally make (i.e., express) that protein. Heterologous polypeptide or heterologous protein thus refers to the fact that the transferred DNA coding for a polypeptide or protein such as insulin or proinsulin was initially cloned from or derived from a different cell type or a different species than the recipient. For example, the gene encoding the proinsulin precursor can be made synthetically and then transferred into the host organism, which as native organism does not produce that polypeptide or protein. Therefore, the genetic material encoding for the polypeptide or protein is added to the recipient cell by recombinant cloning techniques known in the art. The ge- netic material that is transferred for the heterologous expression must be within a format that encourages the recipient cell to express the recombinant DNA as open reading frame (ORF) to synthesize a protein, i.e., it is put in an expression vector.

A "polypeptide" refers to a single linear chain of amino acids. A "protein" refers to a pol- ypeptide, which has the ability to form into a specific conformation. Thus the terms polypeptide and protein can generally be used interchangeably for polypeptides of a specific length.

The term "recombinant DNA" refers to the form of artificial DNA such as a synthetical DNA or cDNA encoding a proinsulin precursor that is created through the introduction of the DNA into an organism such as E. coli for the purpose of expression of the polypeptide or protein encoded by the recombinant DNA. A recombinant protein thus is a protein that is derived from the recombinant DNA by expression of the recombinant DNA in the host cell. The recombinant DNA techniques required for transferring the recombinant DNA into a host organism and expression of the recombinant DNA to yield the recombinant protein in the organism are known to the one skilled in the art. An "insulin precursor" refers to a molecule, which comprises, contains or is homologue to insulin chains A and B, comprising analogues, derivatives and fragments thereof. A human insulin precursor refers to a polypeptide, which contains or is homologous to human insulin chains A and B, comprising analogues, derivatives and fragments thereof.

A "correctly folded" human insulin precursor refers to a molecule wherein the human insulin precursor has the conformation and disulfide bridges as found in a natural, biologically active human insulin, i.e., the disulfide bridges between a) A-6 and A-l 1, b) between A-7 and B-7 and c) between A-20 and B-19 are formed.

In a first aspect, the present invention provides an amino acid sequence, which comprises at the N-terminus the amino acid sequence consisting of methionine, alanine, lysine and arginine (MAKR) and which further comprises an amino acid sequence of a proinsulin precursor. The N-terminal sequence MAKR has the function of a helper sequence, which promotes the expression of the recombinant DNA encoding the polypeptide of the subject invention and which promotes stability of the polypeptide after recombinant expression in a host organism such as E. coli. Thus, as previously shown for the helper sequence MAK, the helper sequence MAKR likewise leads to an increased yield of protein after isolation, purification and processing of the recombinant polypeptide in order to obtain correctly folded insulin.

Preferably, the N-terminal helper sequence is located directly at the N-terminus of an insulin precursor. The insulin precursor preferably comprises the A- and B-chains of human proinsulin. Also described herein is an insulin precursor in which the A- and B-chains of human proinsulin are separated by a single amino acid or by an amino acid sequence consisting of 2 to 35 amino acids. Most preferably, the A- and B-chains are separated by the human C-peptide. In the most preferred embodiment, the human insulin precursor is human proinsulin and thus consists of the B-chain at its N-terminus followed by the C- peptide, which precedes the A-chain at its C-terminus. A concrete example of a polypep- tide having the helper sequence MAKR at its N-terminus and followed by the amino acid sequence of human proinsulin is given by SEQ ID NO 1 : MAKRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGG- PGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

The polypeptide of the present invention may further comprise a sequence, which promotes the purification of the recombinant polypeptide. A His-tag is preferably used for purification of a recombinant polypeptide. The term His-tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least four histidine (His) residues. The prevalently used His-tag consists of six histidine residues and is thus also known as hexa histi- dine-tag or 6xHis-tag. Such a His-tag can be used for affinity purification of the tagged recombinant protein, e.g. after expression in E. coli. Various purification kits for histidine- tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Ma- cherey-Nagel and others.

Different organisms often have different codon specificity to encode a single amino acid. As a consequence, it is preferred in the subject invention that the codon usage of the nucle- otide sequence according to the present invention is adapted for the expression in the respective organism (E.L. Winnacker, Gene und Klone, Verlag Chemie, 1985, 224-241, Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nakamura, Y., Gojobori, T. and Ikemura, T. (2000) Nucl. Acids Res. 28, 292). For example, the DNA encoding the polypeptide comprising the helper sequence and the hu- man proinsulin sequence of the present invention is preferably adapted for expression in the specific organism such as E. coli. Hence, in a preferred embodiment of the invention, the encoding DNA is adapted according to the codon usage of the host organism E. coli.

The subject invention thus relates to an isolated nucleic acid encoding a polypeptide hav- ing the helper sequence MAKR at its N-terminus and further comprising an amino acid sequence of an insulin precursor. The isolated nucleic acid preferably is DNA. The invention further encompasses a nucleic acid sequence which is complementary to the nucleotide sequence encoding a polypeptide having the helper sequence MAKR at its N-terminus and further comprising an amino acid sequence of an insulin precursor. The invention fur- ther encompasses an isolated nucleic acid which hybridizes under mild conditions to a nucleotide sequence, which is complementary to the nucleotide sequence encoding a polypeptide having the helper sequence MAKR at its N-terminus and further comprising an amino acid sequence of an insulin precursor. A concrete example of such a nucleotide sequence, which preferably is DNA is given by SEQ ID NO 2:

20 40 60 atggcgaaac gctttgtgaa ccagcatctg tgcggcagcc atctggtgga agcgctgtat

80 100 120 ctggtgtgcg gcgaacgtgg ctttttttat accccgaaaa cccgtcgtga agcggaagat

140 160 180 ctgcaagtgg gccaggtgga actgggtggt ggtccgggtg cgggtagcct gcaaccgctg

200 220 240 gcgctggaag gcagcctgca aaaacgtggc attgtggaac agtgctgcac cagcatttgc

260

agcctgtatc agctggaaaa ctattgcaac taa

In a further embodiment of the invention the amino acid sequence of the insulin precursor is encoded by the human proinsulin gene sequence or one or more fragments thereof. SEQ ID NO: 3 shows the naturally occurring coding sequence of human proinsulin, wherein the shown sequence is devoid of introns. The complete preproinsulin coding DNA sequence is according to GenBank Acc. No. J00265.1. Thus, the subject invention relates to an isolated nucleic acid encoding a polypeptide having the helper sequence MAKR at its N-terminus and further comprising an amino acid sequence of an insulin precursor encoded by SEQ ID NO: 3 or one or more fragments thereof. SEQ ID NO 3 is:

20 40 60 atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg gggacctgac

80 100 120 ccagccgcag cctttgtgaa ccaacacctg tgcggctcac acctggtgga agctctctac

140 160 180 ctagtgtgcg gggaacgagg cttcttctac acacccaaga cccgccggga ggcagaggac 200 220 240 ctgcaggtgg ggcaggtgga gctgggcggg ggccctggtg caggcagcct gcagcccttg 260 280 300 gccctggagg ggtccctgca gaagcgtggc attgtggaac aatgctgtac cagcatctgc

320

tccctctacc agctggagaa ctactgcaac tag

The DNA of the invention may be obtained by standard procedures known in the art from cloned DNA, e.g., a DNA "library", by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA or fragments thereof, purified from the desired cell. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.

For the expression of the polypeptide of the present invention, the DNA encoding the polypeptide is thus incorporated by standard cloning techniques into an expression vector. The expression vector provides all elements necessary for expression of the recombinant poly- peptide in the heterlogous host. Suitable expression vectors are commercially available and include standard expression vectors for expression in E. coli such as pQET7 available from QIAGEN in which the gene encoding the recombinant protein is expressed under control of the T7 promoter. Transformation of the host cell by the expression vector can be achieved as described by Sambrook et al., Cold Spring Harbor Laboratory Press, 1998 and as known to the one skilled in the art. Suitable E. coli strains are commercially available and include various strains derived from E. coli BL21 such as E. coli C41, commercially available from Lucigen.

The host organism, preferably a prokaryotic cell, most preferably an E. coli cell is trans- formed by a DNA which is modified in a manner as described above and which encodes for the recombinant polypeptide of the present invention, by standard cloning techniques such as transformation of electro competent cells or chemically made competent cells. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered polypeptide may be controlled. A preferred expression system is under control of the T7 promoter of E. coli and induced by the presence of IPTG (isopropyl β-D-l-thiogalactopyranoside).

The resulting heterologous host is then cultivated in a suitable medium. Suitable liquid media for growing the host organism include synthetic media, full or half media. Media for cultivation of E. coli include Luria Broth (LB), 2xYT or, in a particularly preferred embodiment of this invention, a fully synthetic medium based on a phosphate buffer, a nitrogen source like ammonium chloride, a carbon- and energy source like glucose or glycerol, trace elements, and an amino acid supplement to enhance growth (Korz, DJ et al. (1994), J. Biotech. 39, 59-65). Suitable conditions for cultivation are adapted to the organism according to standard procedures known to the skilled person. These include inocculation of the growth media with a starter culture and incubating the cells at a temperature between 25 °C and 42 °C. When grown in a flask, the concentration of dissolved oxygen is enhanced by vigorous shaking. In a fermenter, ample air supply is necessary. The pH of the culture should be kept between 5 and 8. At appropriate cell densities, expression of the recombinant protein is induced by addition of isopropyl-B-D-galactopyranosid (IPTG) or lactose. After sufficient growth of the heterologous organism, the cells are harvested e.g. by filtration or centrifugation and then disintegrated to further isolate the recombinant protein from the broken cells. Disintegration of cells can be achieved by high pressure homogenisation using a high pressure cell such as a french press cell. Other methods of disintegration of cells include enzymatic treatment with lysozyme and sonication.

In E. coli the recombinant polypeptide of the present invention is usually present in form of insoluble inclusion bodies. Thus, the inclusion bodies have to be isolated from the broken cells by washing the broken cells with mild detergents such as Tween 20 or Triton X 100 and with low concentrations of urea, preferably up to 2 M. The recombinant protein is then solubilised and renaturated under conditions, which allow formation of correct cystine bridges and refolding of the recombinant protein to its native conformation. Accordingly, the process step of subjecting the isolated polypeptide to a folding process, which permits correct folding of the insulin precursor, preferably includes the formation of correct cystine bridges. Suitable conditions include the choice of an appropriate buffer and an appropriate pH for isolation and refolding of the recombinant protein as known in the art (Qiao, ZS et al. (2003), J. Biol. Chem. 278, 17800-17809; Winter, J et al. (2002), Anal. Biochem. 310, 148-155).

To obtain active insulin as product, the correctly folded insulin precursor has to be processed by enzymatic cleavage. Thus, the present invention further relates to a process for obtaining a correctly folded insulin-precursor, wherein the insulin precursor is processed by enzymatic cleavage in order to release the active insulin product. Enzymatic cleavage is achieved preferably by the proteases trypsin and carboxypeptidase B. Enzymatic cleavage can be achieved in separated process steps, in which in a first step the helper sequence MAKR and most of the C-peptide, or any polypeptide located between the A and B chains, are released by incubation with trypsin, which selectively cleaves after the amino acids lysine and arginine, and the arginine at position B31 is trimmed off by carboxypeptidase B in a second step. In another embodiment of this invention, both enzymes are used simultaneously in a single process step. Thus, the helper sequence of the present invention has the additional advantage that it can easily be cleaved off by enzymatic processing due to the presence of the amino acid arginine at position 4 of the helper sequence. The original fear that trypsin selectively cleaves after the amino acid lysine at position 3 of the MAKR sequence so that a single arginine would be left attached to the insulin turned out to be groundless. In contrast, it could be shown that enzymatic cleavage of the MAKR helper sequence was much more complete than with the MAK helper sequence. Conditions for the processing of proinsulin to insulin are known to those skilled in the art (Jonasson, P. et al. (1996), Eur. J. Biochem. 236, 656-661 ; or as described in EP 0 367 161). Efficient enzymatic processing requires a slightly alkaline pH, which is preferably between 7,5 and 9, a temperature preferably between 4 °C and 37 °C, the presence of divalent cations such as calcium and/or magnesium and incubation times preferably between 15 min and 5 hours. The molar ratio of enzymes to proinsulin is preferably between 1 : 100 and 1 : 10000. The processed and correctly folded Insulin is then purified in order to obtain the native product. Purification of the insulin product can be achieved by affinity chromatography, anion or cation exchange chromatography, or by reversed phase chromatography according to procedures known in the art. The purified insulin can then be crystallized and/or lyophi- lized according to standard procedures.

The process steps as shortly described above are known from the prior art as listed above and can be adapted without inventive skill.

A method for the production of insulin from bacterial culture such as E. coli thus generally include the steps of:

a) cloning of the DNA sequence encoding the N-terminal MAKR sequence and the insulin precursor into a suitable expression vector,

b) transforming a suitable organism for expression of the recombinant protein with the expression vector,

c) fermentation of the microorganism,

d) disintegration of cells,

e) isolation of inclusion bodies,

f) solubilization and renaturation of the insulin precursor,

g) incubation of the insulin precursor to allow formation of cystin bridges and folding of the insulin precursor,

h) concentration of the folded insulin precursor,

i) processing the insulin precursor with trypsin and carboxypeptidase to yield active insulin,

j) purification and crystallisation, and

k) lyophilisation of the active insulin.

It is within the knowledge of the skilled person that some of the steps may be modified or that further steps may be included such as further purification steps including chromatographic purification of the processed insulin. The method steps as listed above will now be described in further detail by the following examples.

The examples describe preferred embodiments of the invention and are thus only intended for illustration purposes and are not intended to limit the subject invention in any way. As known to the skilled person, various modifications might be made to the described embodiments, without departing from the scope of the invention as defined by the scope of the subject claims. With regard to the methods as outlined below, i.e. the methods with regards to the expression of the polypeptide of the present invention in an heterologous organism, the methods for isolation of the recombinant protein from inclusion bodies, the methods of purifying the recombinant protein and subsequent refolding and processing, it will be understood that minor modifications in the protocols may be made without departing from the spirit or scope of the invention, i.e., with regard to the choice of solvents, buffers, detergents, denaturants, proteolytic enzymes, separation methods and chromatographic me- dia. Such minor modifications are well within the knowledge of the skilled artisan and are intended to fall within the scope of the appended claims.

Examples Example 1

Expression of pMAKR-PI in E. coli grown in a fermenter

The plasmid pMAKR-PI is derived from an expression vector designed for the expression of a recombinant DNA in E. coli. The plasmid contains a gene according to the subject invention and given by SEQ ID NO: 2 encoding human proinsulin and containing a sequence encoding the N-terminal sequence methionine-alanine-lysine-arginine (MAKR) located at the N-terminus of the insulin precursor. The plasmid was transformed into E. coli C41 cells and grown on 2xYT solid medium containing kanamycin for selection of transformed cells. 300 cells were obtained on a petri dish. The plasmid was used for expression in a fermenter with 1 1 working volume. Cells were grown in a defined media (Korz, DJ et al. (1994), J. Biotech. 39, 59-65), supplemented with amino acids. Cells were grown at 37 °C. Dissolved oxygen was kept above 20 % saturation, pH was kept constant by the addition of aqueous ammonia whenever needed. When the optical density OD₆oo reached a value of 5, protein expression was induced with IPTG. After 4 more hours, the optical density had reached 30, corresponding to a dry cell weight of 9 g/1.

Cells were harvested and disintegrated by enzymatic treatment with lysozyme followed by sonication. Inclusion bodies were isolated according to standard procedures. Pre-purified inclusion bodies were dissolved in 50 mM glycine buffer at pH 9.5 and solubilised by adding solid urea to a final concentration of 7.5 M. Moreover, 100 mM beta-mercaptoethanol was added to the solution to provide a strongly reducing environment. Incubation was conducted for 1 hour at 20-37°C using slight agitation; subsequently, insoluble cellular debris was discarded.

The supernatant/filtrate was purified directly by column-based anion exchange chromatography. During the washing of the loaded column using 50 mM glycine buffer at pH 9.5, the concentrations of urea and beta-mercaptoethanol were lowered to 5M urea and 5 mM beta-mercaptoethanol. Fully denatured and reduced proinsulin was eluted using a linear gradient of NaCl.

The elution fractions were diluted to a protein concentration of 0.1 - 1,5 g/1 in refolding buffer, which contained 50 mM glycine, pH 10.5, and urea adjusted to a final concentration of 1 M. Refolding was performed by standard procedures. Misfolded insulin precursor molecules were precipitated by mild acidification and incubation for 1 hour in the cold under continuous stirring. Precipitates were separated by paper filtration.

The pH of the filtrate obtained from the filtration step was adjusted to 3.5 using 1 M HC1 and loaded directly onto a pre-equilibrated cation exchange column. At this pH, proinsulin is positively charged and can be eluted by reversing the charges via a rapid pH change. The combined elution fractions were subjected directly to anion exchange chromatography for a final purification step. The column-bound proinsulin was washed with 20 mM Glycin pH=8.5 and eluted with 150 mM NaCl/20 mM Glycine pH=8.5.

At this point, the proinsulin was judged to be 99 % pure by analytical reverse phase high performance liquid chromatography.

The eluted material was directly subjected to enzymatic processing with trypsin and car- boxypeptidase B. Fig. 1 shows a MALDI-TOF analysis for enzymatically processed MAKR- insulin precursor. The MALDI-TOF analysis shows a single species of human insulin at 5804 d in the peak fraction. No significant amounts of by-products could be detected.

Comparative Example 1

Expression of pMAK-PI in E. coli grown in a fermenter

The plasmid pMAK-PI differs from the plasmid pMAKR-PI by the DNA sequence encoding for the N-terminal sequence preceding the insulin precursor. The encoded polypeptide has the N-terminal sequence Met-Ala-Lys-Proinsulin and thus differs from the encoded polypeptide of Example 1 in that the amino acid arginine is missing in the N-terminal helper sequence.

Expression of plasmid and isolation, purification and processing of recombinant protein was performed as in Example 1.

The resulting protein fraction was analyzed by MALDI-TOF. A single peak was detected at 6010 d, which could be shown to result AK-lnsulin (the methionine of MAK already gets cleaved off by the bacteria). Therefore, enzymatic cleavage by trypsin turned out to be inefficient. The protein fraction was then incubated with an excess of trypsin and the resulting recombinant protein analyzed by MALDI-TOF. The resulting chromatography is shown in Fig. 2. Fig. 2 shows a MALDI-TOF analysis for the enzymatically processed MAK-insulin precursor. In addition to insulin at 5811 d, a considerable amount of unwanted by-products such as des30 insulin was generated (peak at 5710 d).

As a result it could be shown by the above Experiments that the polypeptide according to the subject invention, i.e. a polypeptide having the helper sequence MAKR at the N- terminus of an insulin precursor, firstly leads to an increased cell growth which is comparable to the cell growth achieved with the helper sequence MAK shown previously and which consequently leads to higher amounts of total cellular protein which can be used for the manufacture of insulin.

Secondly, due to the absence of unwanted by-products, the polypeptide of the subject invention leads to a higher relative amount of polypeptide comprising the insulin precursor when compared to the total cellular protein. The invention thus leads to an increased ratio of the polypeptide according to the subject invention to the total cellular protein. As a con- sequence, the higher starting amount of insulin precursor enables to yield higher amounts of active insulin after purification and processing of the insulin precursor.

Example 2 Expression of pMAKR-PI in E. coli grown in shaking cultures

The plasmid used in Example 1 was transformed into E. coli C41 cells and grown on 2xYT solid medium containing kanamycin for selection of transformed cells. 5ml of 2xYT medium containing kanamycin were inoculated with a single colony and incubated overnight at 37°C under vigorous shaking. 400ml prewarmed 2xYT medium containing kanamycin were inoculated with the overnight culture and further incubated at 37°C under vigorous shaking. When the culture reached an optical density OD₆Q_O of about 0.7, expression was induced by adding IPTG to a final concentration of 0.8 mM. Growth was checked by measuring the optical density and samples were taken in periodic intervals.

Isolation, purification and processing of recombinant protein was performed as described in Example 1.

Comparative Example 2

Expression of pMAK-PI in E. coli grown in shaking cultures

The plasmid of comparative Example 1 was used for expression in shaking cultures under the same conditions as described for Example 2. The cell density at induction was the same as in Example 2. Growth and expression conditions of E. coli cells transformed with pMAK-PI were as described above for Example 2.

It could be shown by a comparison of Example 2 with comparative Example 2 that the findings made above by the comparison of Example 1 with comparison Example 1 for the expression of a polypeptide according to the subject invention in a fermenter equally apply for E. coli cultures grown in shaking cultures.

As a result, it could be shown by the Examples according to the present invention that expression of a gene encoding a polypeptide containing a insulin precursor and the N- terminal helper sequence MAKR leads to an increased growth rate of the heterologous host comparable to the growth shown previously for the construct MAK-proinsulin. By increasing the growth rate of the heterologous organism, the D A according to the subject invention encoding for the helper sequence MAKR and human proinsulin leads to an increased yield of recombinant protein. Accordingly, the subject invention enables to yield higher amounts of active insulin after purification and processing the proinsulin precursor due to higher starting amounts of recombinant protein. As a consequence, it could be shown by the present invention that the growth rate of a heterologous organism expressing a gene encoding a recombinant polypeptide comprising an insulin precursor and gene expression said recombinant polypeptide not only depends on the N-terminal amino acid, but can be significantly increased by a helper sequence accord- ing to the subject invention.

Moreover, compared to the comparative construct having the helper sequence MAK- proinsulin, in which the amino acid arginine of the helper sequence is absent, processing and enzymatic cleavage is much more efficient, leading to fewer amounts of unwanted by- products and to an efficient and complete cleavage of the helper sequence.

In summary, it could be shown by the above experiments that expression of an insulin precursor including the N-terminal helper sequence MAKR reduces the generation of byproducts and thereby further increases the amount of insulin product after processing.

Claims

24 Claims

1. A polypeptide comprising,

i. a first peptidyl fragment consisting of the N-terminal amino acid sequence methionine, alanine, lysine and arginine (MAKR), and

ii. a second peptidyl fragment, which is an insulin precursor.

2. The polypeptide of claim 1 , wherein the insulin precursor comprises insulin chains A and B.

3. The polypeptide of claims 1 or 2, wherein the insulin precursor is human proinsulin.

4. The polypeptide of any of the previous claims, further comprising a sequence, which is capable of binding to a chromatographic matrix, preferably, an affinity tag.

5. The polypeptide of claim 4, wherein the affinity tag is a His-tag.

6. The polypeptide of claims 1 , 2 or 3, consisting of the amino acid sequence of SEQ ID NO: 1.

7. An isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of any of claims 1 to 6.

8. The isolated nucleic acid of claim 7, wherein said nucleic acid is DNA.

9. The isolated nucleic acid of claims 7 or 8, which employs a plurality of alternative codons to those present in the naturally occurring wild-type human proinsulin coding sequence, said alternative codons causing no amino acid changes from wild- type human proinsulin, wherein at least a portion of said alternative codons are more preferred for usage in bacterial cells. 25

10. The isolated nucleic acid of claim 9, which consists of the nucleic acid sequence of SEQ ID NO: 2.

11. A recombinant cell containing the nucleic acid of any of claims 7 to 10.

12. The recombinant cell of claim 11, which is Escherichia coli.

13. A method of producing a polypeptide comprising growing a recombinant cell containing the nucleic acid of any of claims 7 to 10 such that the encoded polypeptide is expressed by the cell, and recovering the expressed polypeptide from the cell.

14. A method of producing insulin which comprises the steps of

a. expressing the polypeptide according to any of claims 1 to 6 in Escherichia coli,

b. isolating the polypeptide from the recombinant cell,

c. subjecting the isolated polypeptide to a folding process which permits correct folding of the insulin precursor, and

d. subjecting the polypeptide to enzymatic processing to yield active insulin.