WO2012098009A1 - Polypeptide chimérique comprenant une protéine membranaire et un précurseur de l'insuline - Google Patents

Polypeptide chimérique comprenant une protéine membranaire et un précurseur de l'insuline Download PDF

Info

Publication number
WO2012098009A1
WO2012098009A1 PCT/EP2012/000266 EP2012000266W WO2012098009A1 WO 2012098009 A1 WO2012098009 A1 WO 2012098009A1 EP 2012000266 W EP2012000266 W EP 2012000266W WO 2012098009 A1 WO2012098009 A1 WO 2012098009A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
chimeric polypeptide
polypeptide
amino acid
acid sequence
Prior art date
Application number
PCT/EP2012/000266
Other languages
English (en)
Inventor
Sophia Ponomarenko
Original Assignee
Glucometrix Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glucometrix Ag filed Critical Glucometrix Ag
Publication of WO2012098009A1 publication Critical patent/WO2012098009A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention is concerned with a chimeric polypeptide comprising an insulin precursor, wherein the insulin precursor is coupled to a membrane protein.
  • the invention is further concerned with a nucleic acid coding for the chimeric polypeptide.
  • the invention further relates to a method of producing a recombinant protein such as insulin, wherein a nucleic acid coding for a chimeric polypeptide is expressed in a heterologous host.
  • the method of the invention has the advantage that the recombinant protein can be isolated from the cell membrane of the heterologous host and thus is not produced in inclusion bodies.
  • the isolated recombinant protein further contains intracellularly formed disulfide bridges.
  • the insulin precursor is transformed in vivo into a prefolded intermediate, thus allowing easy processing of the recombinant protein into the correct three-dimensional conformation.
  • 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) 285 million people suffered from diabetes mellitus in 2010. Insulin therapy 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.
  • WHO World Health Organization
  • Human insulin is a hetero dimer and consists of two separate peptide chains, which are the A-chain (iA) of a length of 21 amino acids and the B-chain (iB) of a length of 30 amino acids. These peptide chains are joined together by a characteristic pattern of disulfide bridges. Two disulfide bridges connect the A-chain and the B-chain (Cys-A7— Cys-B7 and Cys-A20— Cys-B19). A third disulfide bridge is formed within the A-chain connecting cysteine residues A6 and Al 1.
  • insulin is produced as a single pre- proinsulin polypeptide which contains an N-terminal signal sequence and proinsulin in which the prospective A- and B-chains are linked together by the C-peptide.
  • Development of native insulin from proinsulin molecule involves formation of correct disulfide bridges and protein folding into the correct three-dimensional conformation. Proinsulin is then processed proteolytically, which results in the cleavage of the C-peptide and release of insulin as active hormone.
  • insulin as a medicine was produced from animal sources such as bovine and porcine pancreatic preparations. Insulin from animal sources, however, differs from human insulin and thus may elicit an adverse immune reaction of patients. Due to the enormous demand for insulin as medicament, biosynthetic human insulin has been manufactured for widespread replace hormone therapy clinical use using recombinant DNA technology for more than 20 years. Due to high rate of synthesis and rapid growth, the primary source for the manufacturing of biosynthetic recombinant proteins such as insulin is its production in prokaryotic organisms such as Escherichia coli (E. coli). To date more than 95 % of all diabetes patients receive the recombinant insulin product.
  • E. coli Escherichia coli
  • inclusion bodies Upon expres- sion 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 solu- bilise the protein.
  • the isolated protein must subsequently be refolded in vitro into the na- tive conformation. This usually requires the formation of the correct disulfide bridges (also synonymously referred to as cystine bridges), which were usually not formed in the cytoplasm of prokaryotes.
  • disulfide bridges are generally essential in maintaining the native conformation and biological activity of the protein molecule.
  • it is necessary to treat the isolated recombinant protein in vitro under conditions that allow extra- cellular formation of disulfide bridges and folding of the protein into its native conformation. This process is generally called renaturation.
  • renaturation of recombinant protein is a time and cost consuming process and often leads to unsatisfactory results caused by the formation of incorrect disulfide bridges.
  • the incorrect formation of disulfide bridges slows down the process of the recombinant protein to fold into its native conformation. Incorrectly folded protein molecules (isoforms) cannot be converted or processed into its active form, and in consequence incorrect formation of disulfide bridges results in decreased yield of active product and its contamination with isoforms.
  • recombinant protein containing one or more disulfide bridges in their native structure and being overexpressed in a prokaryotic organism such as E. coli is usually unable to fold intracellulary into its native structure.
  • recombinant protein is often toxic to the cell.
  • the cell system thus prevents its synthesis in several ways such as reducing the transcription or translation of the heterologous gene, unspecific enzymatic degradation of the recombinant protein, or formation of large insoluble protein aggregates such as inclusion bodies. In consequence, these cell mechanisms lead to decreased yields of protein upon recombinant expression.
  • Figure 1 shows a map of a plasmid containing a DNA sequence coding for the chimeric polypeptide according to a specific embodiment of the invention.
  • Figure 2 shows a schematic protein model of a constructed chimeric polypeptide of the invention.
  • Figure 3 shows an SDS-polyacrylamide gel electrophoresis (PAGE) analysis and western blot of a chimeric polypeptide of the invention synthesized using an "in vitro protein translation system".
  • PAGE SDS-polyacrylamide gel electrophoresis
  • Figure 4 shows an SDS-PAGE analysis and western-blot using an anti-insulin antibody for detection of a chimeric polypeptide of the invention after recombinant expression.
  • Figure 5 shows the cell localization of a chimeric polypeptide after recombinant expression according to the method of the invention.
  • Figure 6 shows a western blot analysis confirming disulfide bond formation in a chimeric polypeptide after recombinant expression according to the method of the invention.
  • Figure 7 shows recombinant expression of the chimeric polypeptide of the invention with and without coexpression of subunit b of E. coli ATP synthase (F 0 b).
  • Figure 8 shows an analysis confirming A20-B19 disulfide bond formation in the insulin precursor obtained by the method according to an embodiment of the invention.
  • Figure 9 shows an analysis confirming semifolding of an insulin heterodimer obtained by the method according to an embodiment of the invention
  • the technical problem of the present invention is to increase the yield of active protein obtained after recombinant expression, isolation and processing. It is another object of the present invention to enhance the yield of active insulin after recombinant expression of an insulin precursor. Moreover, it is an object of the present invention to obtain a recombinant polypeptide, in particular a recombinant insulin precursor, after expression of the polypeptide in a recombinant cell, in particular a prokaryotic host, which has correctly formed disulfide. Thus, it is another object of the present invention to provide a process for the pro- duction of a recombinant polypeptide such as an insulin precursor, wherein correct disulfide bridges are formed in vivo.
  • recombinant expression of the chimeric polypeptide results in the intracellular formation of disulfide bonds (synonymously designated as disulfide bridges or cystine bridges) within the chi- meric polypeptide.
  • a DNA coding for the chimeric polypeptide thus can be expressed in a heterologous host and subsequently isolated from the host, wherein the isolated chimeric polypeptide has correct disulfide bridges, wherein "correct" means that disulfide bridges are present as in the native form of the polypeptide.
  • the subject invention has the advantage that the semifolded chimeric polypeptide can be easily transformed in vitro into its native conformation after recombinant expression of the chimeric polypeptide due to the presence of intracellularly formed disulfide bridges.
  • the solution to the technical problems is the provision of a chimeric polypeptide, an isolated nucleic acid coding for the chimeric polypeptide and a method for producing the recombinant chimeric polypeptide (rCP) having the features as described in the following.
  • the present invention provides a chimeric polypeptide, comprising
  • a first peptidyl fragment comprising an amino acid sequence of a membrane protein
  • a second peptidyl fragment which is an insulin precursor.
  • the insulin precursor comprises insulin chains A and B.
  • nucleic acid such as DNA, comprising a nucleotide sequence coding for a polypeptide, comprising
  • a first peptidyl fragment comprising an amino acid sequence of a membrane protein
  • a second peptidyl fragment which is an insulin precursor.
  • the invention further provides a method of producing a recombinant chimeric polypeptide, comprising the steps of
  • a first peptidyl fragment comprising an amino acid sequence of a membrane protein
  • the present invention provides a method of producing insulin, comprising the steps a) to g) of
  • g) optionally subjecting the active insulin to a purification, crystallization and/or lyo- philisation step.
  • chimeric polypeptide is defined as including any polypeptide where an amino acid sequence of a first peptidyl fragment such as a membrane protein is coupled (synonymously: fused) to an amino acid sequence of another peptidyl fragment such as an insulin precursor.
  • the coupling is achieved in a manner, which provides for a functional transcribing and translating of the DNA segment and message derived there from, respectively.
  • Coupling of DNA sequences can be achieved e.g. by standard recombinant DNA techniques.
  • heterologous expression means that the protein is experimentally put into a cell that does not normally make (i.e., express) that protein.
  • Heter- ologous polypeptide or heterologous protein thus refers to the fact that the transferred DNA coding for a polypeptide or protein was initially cloned from or derived from a different cell type or a different species from the recipient.
  • the gene encoding the chimeric polypeptide of the invention can be made synthetically and then transferred into the host organism (synonymously designated as heterologous organism or host cell), which as native organism does not produce that polypeptide.
  • the genetic material encoding for the polypeptide or protein can be added to the recipient cell by recombinant cloning techniques known in the art.
  • the genetic material that is transferred for the heterologous expression is generally adapted to 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.
  • ORF open reading frame
  • a "polypeptide” in the context of the invention generally refers to a single linear chain of amino acids.
  • a “protein” generally refers to a polypeptide, which has the ability to form into a specific conformation. Thus the terms polypeptide and protein can usually be used interchangeably for polypeptides of a specific length.
  • recombinant DNA generally refers to the form of artificial DNA such as a synthetic DNA or cDNA encoding a insulin 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 polypeptide or recombinant protein thus is a polypeptide or protein that is derived from the recombi- nant DNA by expression of the recombinant DNA in the host cell.
  • insulin precursor refers to a molecule, which comprises, contains or is homologue to insulin chains A and B, generally comprising analogues, derivatives and fragments thereof, and which can be processed into active insulin.
  • a human insulin precursor refers to a poly- peptide, which contains or is homologue to human insulin chains A and B, generally comprising analogues, derivatives and fragments thereof, and which can be processed into active insulin.
  • a “membrane protein” is a protein molecule that is covalently or non-covalently bound, attached or associated with the membrane of a cell or an organelle.
  • a “transmembrane protein” is a protein that spans the membrane of a cell or an organelle.
  • a "correctly folded" molecule such as a polypeptide or protein such as a “correctly folded” recombinant protein refers to a molecule which has the three dimensional conformation and disulfide bridges as found in the native, biologically active protein. In case of human insulin the native protein has disulfide bridges between cysteine residues in positions: a) A-6 and A-l 1, b) between A-7 and B-7 and c) between A-20 and B-19.
  • the present invention provides chimeric polypeptide comprising,
  • a first peptidyl fragment comprising an amino acid sequence of a membrane protein
  • a second peptidyl fragment which is an insulin precursor.
  • the first peptidyl fragment is generally located at the N-terminus (synonymously N-flank) of the chimeric polypeptide. This fragment preferably includes an amino acid sequence of a transmembrane domain.
  • the first peptidyl fragment has the function to assist transport of the chimeric polypeptide of the present invention into the cell membrane. Due to the presence of the first peptidyl fragment, the chimeric polypeptide is covalently or non- covalently, preferably non-covalently, attached to the cell membrane of a heterologous organism upon recombinant expression. More preferably, it is at least partially incorporated into the cell membrane.
  • the first peptidyl fragment comprises an amino acid sequence of the ATP synthase subunit F 0 c.
  • F 0 c refers to the amino acid sequence of subunit c of the F 0 subcomplex of the ATP synthase (F 0 F1).
  • Subunit F 0 c contains a transmembrane domain and contains a large number of hydrophobic amino acids and is therefore also called a proteolipid.
  • the polypeptide F 0 c folds in a- structure and forms a hairpin-like structure with a turn in its half.
  • the ATP synthase subunit F 0 c is a prokaryotic ATP synthase subunit F 0 c of or a fragment thereof, more preferably of the ATP synthase subunit F 0 c of E. coli or a fragment thereof, but can also be of other origin such as such as the corresponding subunits from bacterial, mitochondrial or chloroplast ATP synthases, or a fragment thereof.
  • the total subunit c from E. coli ATP synthase (EF 0 EF1) consists of 79 amino acids and has the amino acid sequence as deposited under GenBank accession no. AAA24732.
  • amino acid sequence of the ATP synthase subunit F 0 c can further comprise one or more amino acids additions or substitutions.
  • analogues of the ATP synthase subunit F 0 c differing from the native sequence are also comprised by the present invention.
  • a fragment of the ATP synthase subunit F 0 c preferably has a length of least 40 amino acids, more preferably at least 60 amino acids and most preferably at least 75 amino acids.
  • the first peptidyl fragment comprises or consists of the amino acid sequence lMet-75Met of the ATP synthase subunit F 0 c of E. coli.
  • the first peptidyl fragment has the amino acid sequence of SEQ ID NO 1 : 10 30 50
  • the second peptidyl fragment is an insulin precursor. It is generally attached to the C- terminus of the first peptidyl fragment, e.g. to the C-terminus of the polypeptide of SEQ ID NO 1 of the present invention.
  • the insulin precursor comprises the A- and B-chains of proinsulin, most preferably of human proinsulin.
  • the amino acid sequence of human proinsulin is as deposited under GenBank accession no. NP 000198.
  • the insulin precursor further comprises insulin analogs.
  • An insulin analog is a modified form of insulin, different from any occurring in nature, but still available to the human body for performing the same action as the naturally occurring insulin. The modification typically comprises the alteration of one or more amino acids of the insulin sequence.
  • Known and commercially available insulin analogs include insulin lispro (Humalog ® ), insulinaspart (Novorapid ® ), insulin glulisin (Apidra ® ), insulin glargin (Lantus ® ) and Insu- lindetemir (Levernir ® ).
  • an insulin precursor in which the A- and B-chains of proinsulin, such as human proinsulin, are separated by a single amino acid or by an amino acid sequence consisting of 2 to 35 amino acids (C'-peptide).
  • C'-peptide is an amino acid sequence having at least 15 %, preferably at least 20 %, most preferably at least 25 % homology with human proinsulin chain C.
  • the A- and B- chains may be separated by an amino acid sequence having at least 15 %, preferably at least 20 %, most preferably at least 25 % identity with human proinsulin chain C.
  • the A- and B-chains of proinsulin may be separated by proinsulin chain C (C -peptide), preferably human insulin chain C (human C-peptide).
  • the A- and B- chains may be separated by an amino acid sequence having a catalytic function.
  • Suitable catalytic functions include protein folding or formation of chemical bindings such as disulfide bonds.
  • Other catalytic functions include function as molecular chaperone, preferably intramolecular chaperone (IMC), foldase, isomerase, e.g. disulfide isomerase, or folding helper sequence (FHS) and can generally be described as enzymes that assist in folding and support other proteins in their non-covalent folding or unfolding and in their assembly and disassembly. These enzymes thus generally have the function to prevent posttranslational aggregation of the polypeptide chain into non-functional structure.
  • IMC intramolecular chaperone
  • FHS folding helper sequence
  • IMC intramolecular chaperones
  • IMC intramolecular chaperones
  • Molecular chaperones can be defined as a class of proteins that assist correct folding of other polypeptides but are not components of the functional assembled structure as described in: Shinde and Inouye, TIBS, 1993,18: 442-446.
  • IMC are part of the precursors of the target proteins to be folded and in their absence the target protein molecules do not have enough information for proper self-folding. Function and unique features of IMC are described in Inouye, Enzyme, 1991, 45: 314-321).
  • IMC include the propeptides of subtilisin, -lytic protease, carboxypeptidase g and ubiquitin (Shinde and Inouye, TIBS, 1993,18: 442-446).
  • the C -peptide is an amino acid sequence consisting of a fragment of the ATP synthase subunit F 0 c of E. coli. In a more preferred embodiment this sequence consists of the sequence 18Gly-47Pro of the ATP synthase subunit F 0 c of E. coli. As described for the F 0 c subunit for the first peptidyl fragment, this specific peptidyl fragment has a transmembrane part and folds in a- structure and further forms a hairpin-like structure with a turn in its half. It has the amino acid sequence of SEQ ID NO 2:
  • a concrete example of a polypeptide comprising human insulin chains A and B being separated by a fragment of the ATP synthase subunit F 0 c of E. coli is given by SEQ ID NO 3:
  • the chimeric polypeptide of the present invention thus preferably comprises, from N-terminus to C-terminus,
  • the chimeric polypeptide of the present invention may further comprise a sequence, which promotes its purification such as purification from a cell extract or during isolation of the polypeptide as described further below for the method of the subject invention. Purification is preferably achieved by metal affinity chromatography such as nickel or cobalt affinity chromatography.
  • a sequence, which is preferably used for purification of the polypeptide of the subject invention is a His-tag, which is able to bind to metal affinity matrices.
  • His-tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least four histidine (His) residues.
  • a His-tag consists four, five or six histidine residues and is thus also known as tetra histidine-tag (4xHis-tag), penta histidine-tag (5xHis- tag) or hexa histidine-tag (6xHis-tag).
  • 4xHis-tag tetra histidine-tag
  • penta histidine-tag 5xHis- tag
  • 6-xHis-tag hexa histidine-tag
  • His-tag can be used for affinity purification of the tagged recombinant protein, e.g. after synthesis in expression systems including a heterologous host such as E. coli.
  • Various purification kits for histidine-tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel and others.
  • a sequence promoting the purification of the recombinant polypeptide such as a His-tag is preferably located N-terminally or C-terminally of the insulin-precursor.
  • such a sequence is located N-terminally or C-terminally of the sequence separating the A-chain from the B-chain of proinsulin, most preferably at the C-terminus of the C'-peptide or C-peptide.
  • the sequence separating the A- and B-chains of insulin is a fragment of the F 0 c subunit as described above, a His-tag prefer- ably locates near the subunit F 0 c turn and thus would not interrupt a-structure of its transmembrane part.
  • His-tag used in the present invention should be as short as possible in order not to negatively influence function and conformation of the chimeric polypeptide.
  • a His- tag of three or less histidine residues is, however, usually not long enough to provide sufficient binding affinity for binding to the chromatographic matrix.
  • a concrete example of a polypeptide, comprising human insulin chains A and B being separated by a fragment of the ATP synthase subunit F 0 c of E. coli and containing a His- tag of four histidine residues, is given by SEQ ID NO 4:
  • the single fragments of the chimeric polypeptide of the invention such as the first peptidyl fragment and the second peptidyl fragment can be connected by the introduction of one or more amino acids as linker.
  • a linker sequence generally comprises between 1 and 10 amino acids, preferably between 1 and 4, most preferably 1 or 2 amino acids. It is particularly preferred that a linker sequence includes a cleavage site, e.g. a protease cleavage site, such as a single lysine or arginine residue.
  • a cleavage site has the advantage that the first peptidyl fragment can be cleaved in vitro from the second peptidyl fragment by use of a suitable protease such as trypsin.
  • Such a linker sequence can also be introduced between one or more fragments within the second peptidyl fragment.
  • a linker sequence can be introduced N-terminally or C-terminally of the A- and the B-chain, and/or between the A- and B- chains of insulin and/or between the A-chain and the amino acid sequence separating the A- and the B-chain such as a sequence having at least 25 % homology with the human insulin C-peptide.
  • a linker sequence can further be used N-terminally or C- terminally of a His-tag.
  • a single methionine (Met) residue can be introduced between the first and the second peptidyl fragment of the chimeric polypeptide of the invention.
  • a single methionine residue can be introduced, e.g. N-terminally of the A-chain and/or of the B- chain, preferably N-terminally of the A- and the B -chain of the insulin precursor, for chemically cleaving the chimeric polypeptide of the invention at these Met-bonds.
  • Chemical cleavage of methionine bonds is usually achieved with BrCN.
  • methionine bonds can be cleaved enzymatically.
  • linker sequences such as protease cleavage sites or chemical cleavage sites allow cleaving and separation of first and the second peptidyl fragment of the chimeric polypeptide of the invention and of the A- and B-chains of the insulin precursor, thereby resulting in the release of insulin.
  • the A- and B-chains of insulin are the only amino acid se- quences within the chimeric polypeptide of the present invention containing cysteine residues.
  • Other cysteine residues, which are not located in the A- and B-chains of insulin, might lead to formation of incorrect disulfide bridges.
  • Such incorrect disulfide bridges e.g. disulfide bridges between the first and second peptidyl fragment, can impede folding of the chimeric polypeptide into the correct three-dimensional conformation.
  • amino acid sequence arginine-lysine is preferably introduced at the C- terminus of the A-chain, followed by a sequence having at least 25 % homology with the human insulin C-peptide, which might be followed by His-tag.
  • the chimeric polypeptide of the invention has the structure:
  • P' -Met - insulin A-chain - Lys - Arg - C - His-tag - Met - insulin B-chain, wherein P' means the first peptidyl fragment of the invention and C means the peptidyl fragment of an amino acid sequence having at least 25 % homology with the C-peptide of human insulin.
  • P' means the first peptidyl fragment of the invention
  • C means the peptidyl fragment of an amino acid sequence having at least 25 % homology with the C-peptide of human insulin.
  • the chimeric polypeptide as described above can further be coexpressed with ATP synthase subunit b (F 0 b) or a fragment thereof, preferably of the ATP synthase subunit b of E. coli.
  • the F 0 b subunit is a membrane bound protein consisting of 156 amino acids. It has been shown that production of F 0 b enhances expression of re- combinant polypeptides in bacterial cells, especially of membrane proteins. Expression of recombinant F 0 b polypeptides generates intracellular membranes (Arechaga et al., FEBS Letters 2000, 482, 215-219; WO 98/02559).
  • F 0 b peptide leads to formation of intracellular membranes without formation of inclusion bodies, thereby enabling easy recovery and isolation of the recombinant chimeric polypeptide from the recombinant cell.
  • the gene of F 0 b subunit or a fragment thereof is located as in the unc operon (EMBL GenBank ID JO 1549), thus after the stop codon of the gene coding for the chimeric polypeptide of the invention.
  • Figure 2 A schematic representation of such a gene construct according to a specific embodiment of the invention is shown in Figure 2. It could further be shown in the present invention that in- crease in expression of the chimeric polypeptide of the invention can also be achieved by using a fragment of the F 0 b subunit.
  • This fragment of the F 0 b subunit preferably comprises or consists of 72 amino acids forming the first half of the F 0 b subunit. Since this fragment in wild type E. coli cells contains a single cysteine residue encoded by tgc (generally de- signated as Cys20), this codon is preferably changed in the gene construct coding for the chimeric polypeptide of the invention, thereby preventing formation of disulfide bonds between the F 0 b subunit and the insulin precursor, in particular chains A and B of insulin. For example, the tgc codon can be changed to tct coding for serine. Different organisms often have different codon specificity to encode a single amino acid.
  • tgc generally de- signated as Cys20
  • the codon usage of the nucleotide sequence according to the present invention is adapted for the expression in the respective organism.
  • the specific codon usage is described in: 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.
  • the chimeric polypeptide of the present invention is preferably adapted for expression in a prokaryotic organism such as E. coli.
  • the DNA encoding the chimeric polypeptide of the present invention described above is adapted according to the codon usage of the host organism E. coli and especially to the codon usage within the unc operon.
  • the unc operon of E. coli (EMBL GenBank ID JO 1594) consists of eight genes coding for the eight types of subunits of the proton-translocating ATPase.
  • the subject invention further relates to an isolated nucleic acid coding for the chimeric polypeptide of the present invention.
  • the isolated nucleic acid preferably is DNA.
  • the invention further encompasses a nucleic acid sequence, which is complementary to the nucleotide sequence encoding the chimeric polypeptide of the present invention.
  • a specific example of such a nucleotide sequence and coding for an amino acid sequence having the structure: P' -Met - insulin A-chain - Lys - Arg - C - His-tag - Met - insulin B-chain, wherein codon usage of E. coli was considered is given by SEQ ID NO 6:
  • the amino acid sequence of the insulin precursor is encoded by the human proinsulin gene sequence or one or more fragments thereof.
  • the complete proinsulin coding DNA sequence is according to GenBank accession no. J00265.1.
  • the subject invention relates to an isolated nucleic acid encoding a poly- peptide having the first peptidyl fragment as described above at its N-terminus and further comprising an amino acid sequence of the insulin precursor encoded by SEQ ID NO 8 or one or more fragments thereof.
  • SEQ ID NO 8 is:
  • the DNA of the invention may be obtained in silico by standard procedures known in the art, e.g. from cloned DNA such as 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.
  • a rCP such as a recombinant polypeptide including an insulin precursor
  • a rCP such as a recombinant polypeptide including an insulin precursor
  • the present invention relates to a method of producing a recombinant chimeric polypeptide, comprising the steps of
  • the process will be described in the following for the production of a recombi- nant insulin precursor, it is not intended to restrict the method of the present invention to those chimeric polypeptides.
  • the method of the invention offers the advantage that recombinant proteins containing one or more disulfide bridge(s) in its corresponding native conformation can be produced by the method of the present invention under intracellular formation of correct disulfide bridge(s).
  • the chimeric polypeptide is a polypeptide as described above, comprising a first peptidyl fragment, comprising an amino acid sequence of a membrane protein, and a second peptidyl fragment which is an insulin precursor.
  • Process step a) of the method of the present invention refers to the step of growing a recombinant cell containing a nucleic acid coding for the chimeric polypeptide of the invention under conditions such that the encoded polypeptide is expressed by the cell.
  • the DNA, preferably cDNA, encoding the polypeptide is thus generally incorporated by standard cloning techniques into an expression vector.
  • the expression vector provides all elements necessary for expression of the recombinant polypeptide in the heterologous host.
  • Suitable expression vec- tors are commercially available and include standard expression vectors for expression in E. coli such as pET21 available from Novagen or pQET7 available from Qiagen in which the gene coding for 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.
  • Suitable E. coli strains are commercially available and include various strains derived from E. coli BL21 such as E. coli C41 or C43, commercially available from Lucigen.
  • the host organism preferably a prokaryotic cell, most preferably an E. coli cell is transformed by a DNA which is modified in a manner as described above and which encodes for the chimeric polypeptide of the present invention by standard cloning techniques such as transformation of electrocompetent cells or chemically made competent cells.
  • the host organism including the DNA of the present invention can thus also be designated as the recombinant organism or recombinant cell.
  • 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 ⁇ -D-l-thiogalactopyranoside).
  • the resulting heterologous host (the recombinant cell) can be 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 a synthetic medium, 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 as described in Korz, DJ et al. (1994), J. Biotech. 39, 59-65.
  • Suitable conditions for cultivation are adapted to the organism according to standard procedures.
  • Recombinant expression of the chimeric polypeptide of the invention usually leads to yields of more than 5 mg/1 (mg per litre cell culture medium), preferably more than 8 mg/1, most preferably more than 12 mg/1 of recombinant protein.
  • the recombinant protein typically corresponds to more than 2 wt.% of the total cell protein, preferably more than 5 wt.%, most preferably more than 10 wt.% of the total cell protein.
  • the chimeric polypeptide expressed by the recombinant cell typically does not form inclusion bodies but is expressed in the plasmic membrane.
  • the chimeric polypeptide of the present invention is usually attached, associated or incorporated into the plasmic membrane of the host organism. Usually, the chimeric polypeptide of the present invention is partially of completely incorporated into the plasmic membrane of the recombinant cell. In case of gram-negative bacteria such as E. coli, the chimeric polypeptide of the invention is preferably attached, associated or incorporated, most preferably incorporated into the inner membrane of the cell.
  • the method of the subject invention offers the advantage that the rCP expressed by the cell can be isolated from the cell membrane such as the inner membrane. In case of coexpression of the chimeric polypeptide with the F 0 b subunit, the chimeric polypeptide is integrated in plasmic membranes. Moreover, the rCP is usually expressed by formation of dimers.
  • Process step b) of the method of the subject invention refers to the step of recovering the expressed polypeptide from the recombinant cell.
  • Step b) generally includes isolating ("harvesting") the cells by separating the cells from the culture medium, disintegrating the cells and isolating the expressed chimeric polypeptide from the disintegrated cells as explained in the following.
  • the cells can be harvested e.g. by filtration or centrifugation and then disintegrated to further isolate the chimeric polypeptide from the broken cells.
  • Disintegration of cells can be achieved by high pressure homogeni- sation using a high pressure cell such as a french press cell. Other methods of disintegration of cells include enzymatic treatment with lysozyme and sonication. Isolation of the chimeric polypeptide from the cell membrane can be achieved using different methods as described in Arechaga et al., FEBS Letters 2002, p. 189-3; Altendorf et al., 1979; Fillingame, 1976.
  • the rCP isolated from the recombinant cells has one or more correctly formed disulfide bridges and generally is expressed in semifolded form.
  • the invention further relates to a process of producing insulin, comprising steps a) and b) as described above and wherein the second peptidyl fragment is an insulin precursor, and further comprising the additional process steps c) to g), (steps d) and g) optional).
  • additional process steps will be described in the following in more detail. All explanations and descriptions of features concerning the chimeric polypeptide of the invention and the nucleic acid coding for the chimeric polypeptide of the invention also apply to the method of producing insulin as described in the following.
  • Method step c) refers to the step of cleaving the first peptidyl fragment from the second peptidyl fragment.
  • step c) of the method of the present invention thus comprises processing of the rCP to separate the first and the second peptidyl fragment.
  • This process step may optionally further comprise removing one or more of the above mentioned supplementary amino acids or peptidyl fragments. However, removing these supplementary amino acids or peptidyl fragments may also be achieved in an additional process step (step e)), preferably after an optional purification step (step d)).
  • Cleavage can be achieved by chemical separation and/or by enzymatic cleavage.
  • chemical separation and enzymatic cleavage are both applied in one or more process steps to selectively remove specific amino acids or peptidyl fragments.
  • cleavage is achieved by chemical separation of the second peptidyl fragment from the first one using cyanogen bromide (BrCN).
  • BrCN cyanogen bromide
  • a methionine residue is prefer- ably introduced between the first and the second peptidyl fragment, which can be used for separation of these peptidyl fragments.
  • Methionine residues can further be introduced between the C or C peptide and the A- or B-chain of insulin.
  • a particularly preferred construct in which a first methionine residue is introduced between the P' -peptide and the A- chain of insulin and a second methionine residue is introduced between a His-tag located at the C-terminus of the C'-peptide and the B-chain of insulin is shown in Figure 1.
  • methionine residues can be cleaved by proteolytic enzymes such as thiotrans- ferase.
  • proteases include but are not limited to trypsin and/or carboxypeptidase. Preferably both trypsin and carboxypeptidase are used..
  • the preferred carboxypeptidase is carboxypeptidase B.
  • Enzymatic cleavage can be achieved in one process step or in more than one process steps.
  • the chimeric polypeptide obtained from step c) can be incubated with two or more proteases simultaneously or, in the alternative, initially with a first protease such as trypsin or carboxypeptidase and subsequently with a second protease different from the first protease such as carboxypeptidase or trypsin.
  • process step c) involves cleavage of the first peptidyl fragment, whereas the C-peptide or C -peptide is still present in the iAiB heterodimer.
  • the C- or C -peptide is attached to the C-terminus of the insulin A-chain.
  • the His-tag is still present in the insulin intermediate e.g. at the car- boxyterminus of the C-peptide or C -peptide and, thus, preferably is not cleaved in process step c).
  • the His-tag can then be removed after purification (step d)) of the insulin intermediate, e.g. by chemical cleavage with BrCN or, more preferably, by proteolytic treatment as described below (step e)).
  • a first cleavage step may include separation of the first peptidyl fragment P' after the carboxyterminal methionine residue from the second peptidyl fragment by way of BrCN cleavage.
  • the His-tag can be separated from the B-chain in the same step by BrCN cleavage.
  • This embodiment has the additional advantage that the His-tag now has a motile carboxyterminal end, which is not further bound to the B-chain.
  • the His-tag can thus be easily attached to an MAC matrix for purification purposes (as explained further below for process step d)).
  • protease e.g. trypsin cleaves dipeptide R-K (A22-A23) to separate the C'-peptide including the His-tag from the remaining iAiB heterodimer, and the arginine at position A22 may be trimmed off by carboxypeptidase B in the same or in a subsequent step (step e)).
  • both enzymes are used simultaneously in one single step.
  • the present invention has the additional advantage that the first peptidyl fragment can easily be cleaved off by chemical treatment and the remaining polypeptide can then be purified by MAC before removing the C'-peptide and a His-tag, thereby releasing insulin as iAiB heterodimer.
  • Conditions for the in vitro processing of proinsulin to insulin are as described in 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.
  • step c) typically results in the presence of a semifolded heterodimeric protein comprising the A- and B-chains of insulin (and preferably also the C -peptide and a His-tag) including the presence of two disulfide bonds as described further below (crude insulin).
  • the semifolded heterodimeric protein resulting from step c) can optionally be purified, e.g. for separating the crude insulin from other peptidyl fragments.
  • the insulin precursor can subsequently be purified by e.g. metal affinity chromatography (MAC).
  • MAC metal affinity chromatography
  • Purification of the semifolded heterodimeric protein can further be achieved e.g. by chromatographic methods. Suitable chromatographic methods include but are not limited to affinity chromatography, anion or cation exchange chromatography, size exclusion chromatography and gel permeation chromatography according to procedures known in the art. More than one purification method can be applied. Thus, two or more purification steps can be performed subsequently
  • the chimeric polypeptide containing the insulin precursor can be cleaved after isolation in step b), e.g. with cyanogen bromide (step c)) and then diluted to reduce the BrCN concentration for purification by MAC (step d)).
  • the insulin precursor can be attached to the MAC matrix and washed one or more times to further reduce BrCN contamination and contamination with other proteins.
  • the insulin precursor can subsequently be eluted from the chromatographic matrix by standard procedures.
  • the eluted semifolded insulin precursor is about 9 KDa and contains two disulfide bonds.
  • the insulin precursor can then be subjected to e.g.
  • step e) a heterodimeric (iAiB) peptide containing the A- and B-chains of insulin (step e)).
  • the heterodimeric (iAiB) peptide consists of the A- and B-chains of insulin.
  • step f conversion into the native three-dimensional conformation requires formation of correct disulfide bridges and/or folding of the protein (step f)).
  • fold- ing of the protein and formation of one or more correct disulfide bridges can be achieved in one process step or in more than one process step.
  • the second peptidyl fragment of the isolated chimeric polypeptide recovered from the recombinant cell has at least one disulfide bridge formed in the plasmic membrane, i.e. in vivo.
  • the second peptidyl fragment has in its native conformation two or more correctly formed disulfide bridges such as is in the case of an insulin precursor, at least two of these disulfide bridges are typically formed in the plasmic membrane, i.e. in vivo.
  • the insulin precursor produced by the method as described herein contains two disulfide bridges, which are formed in the plas- mic membrane upon expression in the recombinant cell.
  • the polypeptide isolated in step b) has usually two correctly formed disulfide bridges.
  • the A6-A1 1 disulfide bridge and the A20-C19 disulfide bridge can be formed in vivo by posttransla- tional folding due to the presence of the F 0 c subunit. This can probably be explained by the formation of a structural assembly of the rCP having a conformation, which is similar to the native F 0 c dimer.
  • the method of the present invention thus provides the advantage that the insulin precursor isolated form the prokaryotic cell in step b) contains two correctly formed disulfide bridges. Due to the presence of these two correctly formed disulfide bridges, the third disulfide bridge (A7-B7) is formed easily in step f).
  • the insulin precursor isolated from the recombinant cell is already partially folded and thus easily and efficiently further folds into the native three-dimensional conformation.
  • refolding in method of the present invention is much more convenient and efficient and results in greatly improved folding efficiency.
  • the iAiB heterodimer discharges in steps c) to step e) from the rCP in its semifolded form including two disulfide bridges, which helps to build in uncomplicated manner the third A7-B7 disulfide bond in step f).
  • the chimeric polypeptide thus can be treated under conditions that permit the correct formation of the third A7-B7 disulfide bridge and final folding of the semifolded insulin precursor into the native conformation.
  • Suitable conditions include the choice of an appropriate buffer and an appropriate pH for formation of the third disulfide bridge in vitro and finally folding of the polypeptide into its native conformation. Suitable conditions are as described in Qiao, ZS et al. (2003), J. Biol. Chem. 278, 17800-17809 or Winter, J et al. (2002), Anal. Biochem. 310, 148-155; Jia X-Y et al. 2003, ProteinSci. 12, p.2412-2419.
  • the correctly folded insulin is then usually further purified in step g) to obtain the native product.
  • Purification of the insulin product can be achieved e.g. by chromatographic methods. Suitable chromatographic methods include but are not limited to affinity chromatography, anion or cation exchange chromatography, size exclusion chromatography and gel permeation chromatography according to procedures known in the art. More than one purification method can be applied. Moreover, two or more purification steps can be performed subsequently. The purified insulin can then optionally be subjected to a crystallization and/or lyophilisation step to result in the final insulin product
  • the recombinant insulin produced by the method of the present invention can be used for various purposes.
  • the recombinant insulin is used as active pharmaceutical ingredient in insulin medicaments, e.g. for use in diabetes therapy.
  • a specific example according to a preferred embodiment of the method for the production of insulin from bacterial culture such as E. coli thus may include the steps of:
  • Example 1 Construction of cDNA and expression plasmid vector The cDNA coding for the polypeptide having the schematic sequence cP'AC'B as shown in Fig.l plus the N-terminal F 0 b subunit encoded by uncF' was constructed in silico and then chemically synthesized. E. coli codon usage was also considered.
  • Fig. 1 shows the constructed chimeric peptide in the schematic sequence P'AC'B.
  • the polypeptide is about 16 kDa assembled as P' (first peptidyl fragment), A- and B-chains of human insulin separated by C, a homolog of human proinsulin chain C. Arrows show location of Arg residues. M depicts location of methionine residue. Two Cys-Cys bonds are shown in the chimeric peptide.
  • peptide A the nucleotide sequence as described in Ponomarenko, Biochemistry (Moscow) 2006, 71 :9, 1006-12 was used. Met, Lys or Arg residues were introduced to get sites for digestion of the chimeric peptide with BrCN or trypsin subsequently. A His-Tag was introduced for protein purification with MAC.
  • Fig- ure 2 is a schematic representation of the gene construct pBsrG2 showing the assembly of the chimeric polypeptide encoded by the recombinant DNA (rDNA) inserted into the pET expression vector.
  • rDNA recombinant DNA
  • Expression vectors pET type produces recombinant proteins in bacteria under the control of the strong bacteriophage T7 transcription signal.
  • the rDNA was inserted into the pET vector as 0.7 kbp Ndel-Xhol restriction fragment.
  • Promoter, rbs pro- moter and ribosome binding site provided by the pET vector
  • KR-C lysine-arginine- C peptide.
  • the E. coli gene uncF codes for ATP synthase subunit b (F 0 b).
  • the F 0 b is a membrane bound protein consisting from 156 amino acids including the singly cysteine residue (Cys20).
  • Two subunits b build a dimer in en- zyme complex EFoFl .
  • a part of the N-terminal flank of F 0 b locates in the plasmatic membrane near F 0 c and the C-terminal flank binds to the catalytic sector of the ATP synthase.
  • SUb' is the first half of the Fob and has 72 amino acids including the single Cys20 residue.
  • the N-terminal flank of SUb' situates in the plasmatic membrane.
  • Ser Two substitution mutations were done in uncF': in start codon Ctg>Atg; restriction site Asel was removed ATTAAT>TTTAAT.
  • Example 2 Check of protein expression ability by the chimeric construct
  • the designed and chemically synthesized cDNA for chimeric peptide is a fusion product of different human and E. coli genes plus some additional nucleotides for extra amino acids such as linker sequences. Transcription and translation capability of the DNA designed in silico was proved using an in vitro protein translation system.
  • the Vector of Example 1 including the synthetic gene to produce the insulin and containing the chimeric peptide was named pBsrG2.
  • the vector was tested in two different cell-free expression systems: EasyXpress ® Protein Synthesis (Qiagen) and Purexpress ® In Vitro Protein Synthesis Kit (NEB). Experiments were performed according to manufacturer protocols. Protein expres- sion was detected by western blots using anti-His or anti-Insulin antibodies (Fig. 3).
  • Figure 3 shows an SDS polyacrylamide gel (PAAG) of the proteins synthesized using the in vitro protein translation system to detect synthesis of chimeric peptide with human insulin chain A and B in the cell free expression system.
  • Plasmid vector pBsrG2 was used as template for recombinant protein synthesis.
  • Left panel proteins stained with Coomassie Blue R-250 after blotting.
  • Right panel WB with anti-insulin anti bodies.
  • KDa size of the proteins, ins: commercial recombinant human insulin "Berlinsulin ® H Normal”.
  • the figure shows that the chimeric polypeptide of the invention could be synthesized using a cell free expression system.
  • Example 3 Investigation of chimeric peptide expression in bacterial cells
  • E. coli clones were obtained after transformation of competent cells with plasmid vector pBsrG2 according to standard procedures (Sambrook et al, Cold Spring Harbor Laboratory Press) and named TbciEc. Chimeric peptide was produced in the bacterial cells similar to the "cell free translation system". Optimal expression was achieved with E. coli C41 and C43 host cells (Fig. 4). These cells were selected for overproduction of the recombinant protein. Expression of the chimeric peptide in E. coli was induced with 1 mM IPTG. The recombinant protein production was higher at 38 °C than at 25°C. Determination of the protein with Image J (NIH) gave about 12 mg/1 culture (about 10% of total cell protein).
  • Figure 4 shows an SDS-PAGE analysis and western-blot using an anti-insulin antibody for detection of the chimeric polypeptide of the present invention after expression in E. coli and isolation of the recombinant protein.
  • Left panel Western Blot with anti-insulin anti- bodies.
  • Right panel 12% reduced SDS PAAG stained with Coomassie Brilliant Blue R250 after protein blotting. Protein markers are in kDa.
  • figure 4 shows that the chimeric polypeptide of the invention could be recombinantly expressed in E. coli.
  • the chimeric peptide was produced in E. coli C41 or C43 clones which gave better results than BL21 (DE3) or other host cells.
  • the chimeric peptide was isolated from cell membrane. To investigate solubility of the protein some solubilization methods were used.
  • the protein of interest does not form inclusion bodies and mostly locates as dimer in the plas- mic membrane (Fig. 5) of the bacterial cells.
  • Figure 5 shows the cell localization of the expressed chimeric peptide: 12 % SDS PAAG. Left: Western blot with anti-insulin anti bodies. Right: proteins stained with Coomassie Brilliant Blue R250 after blotting. Me: membrane fraction, C: cytozolic fraction.
  • figure 5 shows that the chimeric polypeptide of the invention is expressed in the plasmic membrane of the bacterial cells.
  • the protein was isolated using 2% n-octyl-P-D-glucopyranosid with protease inhibitor by incubation in a thermomixer for 10 min at room temperature and 1 h at 4°C at 500 rpm. The incubation mix was centrifuged for 10 min at 18000 rpm at 4 °C. Supernatant was applied on mini-columns with Sephadex ® 50 and centrifuged for 5 min at 10 000 rpm. The protein was precipitated with 10 % trichloroethanoic acid (TCA) (final concentration 5%) and diluted in non-reduced SDS loading buffer. For western blot analysis anti-insulin antibodies were used. Difference in polypeptide bands was shown in non-reduced 12% PAAG: a part of the chimeric protein moves slowly, which shows that S-S bonds in that protein fraction were reduced after DTT treatment (Fig. 6).
  • Figure 6 shows a western blot with anti-insulin antibodies. 12% non-reduced PAAG. Cells were incubated with CuCl 2 or DTT. Protein standard markers in kDa. This data can be interpreted as that the chimeric polypeptide includes disulfide bridges formed intracellularly.
  • Example 6 The E. coli ATP synthase subunit b enhances expression of the insulin precursor
  • Plasmid vector pciQ contains the same DNA sequence of the first and second peptidyl fragment as expression vector pBsrG2 for expression of the chimeric protein but no DNA for subunit F 0 b.
  • Expression vector pBsrG2 is as shown in Figure 2 containing the gene of the chimeric protein fused with DNA fragment for synthesis of truncated subunit F 0 b.
  • Figure 7 shows recombinant protein synthesis of the chimeric polypeptide of the invention with and without coexpression of subunit b of E. coli ATP synthase (F 0 b).
  • Left panel 12 % SDS-PAAG stained with coomassie brilliant blue R-250. Samples of E. coli cells containing equal amount of the protein of expressed insulin precursor using two expression vec- tors pciQ (without F 0 B subunit, lane 1) and pBsrG2 (with F 0 b subunit, lane 2) in Laemmli Buffer were applied in each slot.
  • Right panel Western blot of the SDS-PAAG with anti- insulin anti-bodies after electrophoresis.
  • Example 7 shows protein synthesis of a recombinant chimeric polypeptide of the F 0 c subunit fused to an insulin precursor. Moreover, recombinant protein synthesis could be significantly enhanced by coexpression of the chimeric polypeptide with E. coli ATP synthase subunit b.
  • Example 7 Control of formation of the A20-B19 disulfide bond in the chimeric polypeptide
  • the chimeric protein builds in vivo disulfide bonds.
  • Variation in digestion methods can help to detect formation a Cys-Cys bond between peptide chains A and B.
  • the largest product is obtained after trypsin digestion of the chimeric peptide. With the presence of the S-S link in A20-B 19 the largest fragment after trypsin digestion should be 91 amino acids (or 82 if the Arg in peptide B was affected). But after reduction of the Cys-Cys bonds the peptides A and B are not further linked resulting in a different peptide digestion pattern (cf. Fig. 1).
  • digestion products of E To find an alteration in distribution of the chimeric peptide, digestion products of E.
  • coli cells were suspended in Buffer TMG and divided in two equal parts. One of them was incubated with 1 mM CuCl 2 and the other with 100 mM DTT for 1 h at 4 °C. The cells were washed in TMG buffer twice by centrifugation, resuspended in trypsin digesting buffer (TdB): 20mM Tris, pH 8, 5mM MgCl 2 , 150 mM KC1 and the en- zyme was added in ratio 1 :500. The enzymatic treatment continued for 15 and 60 min at room temperature. In each slot of the 12% SDS PAAG samples were loaded with equal amount of protein.
  • TdB trypsin digesting buffer
  • Figure 8 shows an analysis confirming A20-B19 disulfide bond formation in the chimeric protein by western blot analysis of the oxidized (CuCl 2 ) or reduced (DTT) chimeric protein.
  • 12% SDS PAAG anti-insulin antibodies 0, 15, 60; time of trypsin digestion in min. SM; recombinant human insulin "Berlinsulin® H Normal" as standard protein marker.
  • the mix was incubated in a thermomixer for 10 min at room temperature and 1 h at 4°C at 500 rpm.
  • the incubation mix was centrifuged for 10 min at 18000 rpm at 4 °C.
  • Clear lysate was centrifuged for 40 min and 30 000 rpm at 4°C.
  • Supernatant was applied on 3 kDa cut-off mini-spin columns and centrifuged for 10 min at 10 000 rpm.
  • BrCN in concentration 60 mM per 10 mg protein was added to the concentrated solution.
  • Trypsin solution in TdB was added in ratio 1 : 1000 to cover the resin with bound protein for 1 h at 37 °C.
  • the largest trypsin and BrCN digestion product of chimeric peptide with S-S link in A20-B19 should be 52 amino acids (or 45 if the Arg in peptide B was affected) and the other peptides must be smaller than 18 amino acids.
  • the chimeric polypeptide with intact Cys-Cys bonds gave the clear signal of a band with size identical of com-bital human recombinant insulin.
  • Active human insulin hormone molecule is a heterodimer consisting of 21 amino acids of peptide A and 30 of peptide B. Supplementary amino acids linked to this insulin molecule for the purposes as described in the present invention should be removed in down stream process. For the removing chemical and enzymatic digestion steps can be used.
  • Figure 9 shows an analysis confirming semifolding of the insulin precursor obtained after expression of the chimeric peptide and processing of the polypeptide by the method as described above.
  • Fig. 9 16% SDS PAAG.
  • Left panel protein staining with Coomassie R-
  • the present invention offers several advantages compared to the prior art processes.
  • the chimeric polypeptide of the invention is expressed in the periplasmic membrane.
  • recombinant expression does not lead to the formation of inclusion bodies.
  • the time and cost consuming process of isolation of inclusion bodies, solubilization of inclusion bodies, in vitro formation of disulfide bridges and complete refolding into the native conformation can be avoided.
  • the recombinant chimeric polypeptide of the invention is post-translationally folded or semi- folded.
  • recombinant expression of the chimeric polypeptide results in the intra- cellular formation of correct disulfide bonds in the chimeric polypeptide. As a consequence, the recombinant polypeptide can be easily processed into the correct three- dimensional conformation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Diabetes (AREA)
  • General Engineering & Computer Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un polypeptide chimérique et un acide nucléique isolé, tel qu'un ADN codant pour le polypeptide chimérique, comprenant i) un premier fragment peptidyle, comprenant une séquence d'acides aminés de la sous-unité F0c de l'ATP synthase et ii) un second fragment peptidyle qui est un précurseur de l'insuline. Dans un mode de réalisation privilégié, le précurseur de l'insuline comprend les chaînes A et B de l'insuline, et de façon davantage privilégiée, est la pro-insuline humaine. L'invention concerne en outre un procédé de production d'un polypeptide chimérique recombinant et un procédé de production d'insuline. Dans le procédé de l'invention, des ponts disulfures sont formés dans le polypeptide recombinant in vivo lors de l'expression dans la cellule recombinante. Le polypeptide recombinant peut être obtenu à partir de la cellule recombinante sous une forme semi-repliée.
PCT/EP2012/000266 2011-01-21 2012-01-20 Polypeptide chimérique comprenant une protéine membranaire et un précurseur de l'insuline WO2012098009A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP11000500 2011-01-21
EP11000500.6 2011-01-21

Publications (1)

Publication Number Publication Date
WO2012098009A1 true WO2012098009A1 (fr) 2012-07-26

Family

ID=44170091

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2012/000266 WO2012098009A1 (fr) 2011-01-21 2012-01-20 Polypeptide chimérique comprenant une protéine membranaire et un précurseur de l'insuline

Country Status (1)

Country Link
WO (1) WO2012098009A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0367161A2 (fr) 1988-11-03 1990-05-09 Hoechst Aktiengesellschaft Procédé pour l'hydrolyse sélective d'une protéine de fusion
WO1996020724A1 (fr) 1994-12-29 1996-07-11 Bio-Technology General Corp. Production d'insuline humaine
WO1998002559A1 (fr) 1996-07-12 1998-01-22 Medical Research Council Surexpression de proteines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0367161A2 (fr) 1988-11-03 1990-05-09 Hoechst Aktiengesellschaft Procédé pour l'hydrolyse sélective d'une protéine de fusion
WO1996020724A1 (fr) 1994-12-29 1996-07-11 Bio-Technology General Corp. Production d'insuline humaine
WO1998002559A1 (fr) 1996-07-12 1998-01-22 Medical Research Council Surexpression de proteines

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
ARCHEGA ET AL., FEBS LETTERS, 2000, pages 215 - 219
ARECHAGA ET AL., FEBS LETTERS, 2002, pages 189 - 3
ARECHAGA ET AL., FEBS LETTERS, vol. 482, 2000, pages 215 - 219
E.L. WINNACKER: "Gene und Klone", 1985, VERLAG CHEMIE, pages: 224 - 241
INOUYE, ENZYME, vol. 45, 1991, pages 314 - 321
JIA X-Y ET AL., PROTEINSCI., vol. 12, 2003, pages 2412 - 2419
JONASSON, P. ET AL., EUR. J. BIOCHEM., vol. 236, 1996, pages 656 - 661
KORZ, DJ ET AL., J. BIOTECH., vol. 39, 1994, pages 59 - 65
M MBIKAY: "A Chimeric Proinsulin-CD5 Protein Expressed in AtT-20 Cells Is Directed to the Cell Surface via the Constitutive Pathway", EXPERIMENTAL CELL RESEARCH, vol. 220, no. 1, 1 September 1995 (1995-09-01), pages 79 - 91, XP055001628, ISSN: 0014-4827, DOI: 10.1006/excr.1995.1294 *
NAKAMURA, Y.; GOJOBORI, T.; IKEMURA, T., NUCL. ACIDS RES., vol. 28, 2000, pages 292
PONOMARENKO S V: "Biochemical characteristics of Escherichia coli ATP synthase with insulin peptide a fused to the globular part of the [gamma]-subunit", BIOCHEMISTRY (MOSCOW), KLUWER ACADEMIC PUBLISHERS-PLENUM PUBLISHERS, NE, vol. 71, no. 9, 1 September 2006 (2006-09-01), pages 1006 - 1012, XP019433465, ISSN: 1608-3040, DOI: DOI:10.1134/S0006297906090094 *
PONOMARENKO, BIOCHEMISTRY, vol. 71, no. 9, 2006, pages 1006 - 12
QIAO, ZS ET AL., J. BIOL. CHEM., vol. 278, 2003, pages 17800 - 17809
SHINDE; INOUYE, TIBS, vol. 18, 1993, pages 442 - 446
TRENDS BIOCHEM., SCIENCE, 2006
WINTER ET AL., J. BIOTECHNOLOGY, vol. 84, 2000, pages 175 - 185
WINTER, J ET AL., ANAL. BIOCHEM., vol. 310, 2002, pages 148 - 155

Similar Documents

Publication Publication Date Title
EP0305500B1 (fr) Procede de purification de polypeptides recombinants
JP5909172B2 (ja) 融合タンパク質の自己タンパク質分解切断による組換えタンパク質の産生
CA2912300C (fr) Procedes pour l'expression de peptides et de proteines
JP4504014B2 (ja) インスリン分泌性ｇｌｐ−１（７−３６）ポリペプチドおよび／またはｇｌｐ−１類似体を生成する方法
JP2019195327A (ja) 枯草菌（ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）において真正で生物活性を有する塩基性線維芽細胞増殖因子を発現させる方法及び手段
CZ290079B6 (cs) Způsob produkce inzulinu a meziprodukt pro tento způsob
US20160168226A1 (en) Process for production of insulin and insulin analogues
ES2754271T3 (es) Polipéptidos escindibles por enteroquinasa
JP7266325B2 (ja) 蛍光タンパク質フラグメントを含む融合タンパク質およびその用途
Ahn et al. Improved recombinant protein production using heat shock proteins in Escherichia coli
US20210230659A1 (en) Leader Sequence for Higher Expression of Recombinant Proteins
US10745457B2 (en) Process for obtaining insulin with correctly formed disulfide bonds
RU2447149C1 (ru) РЕКОМБИНАНТНАЯ ПЛАЗМИДНАЯ ДНК pMSIN4, КОДИРУЮЩАЯ ГИБРИДНЫЙ ПОЛИПЕПТИД - ПРЕДШЕСТВЕННИК ИНСУЛИНА ЧЕЛОВЕКА, ШТАММ BL21(DE3)/pMSIN4-ПРОДУЦЕНТ РЕКОМБИНАНТНОГО ИНСУЛИНА ЧЕЛОВЕКА, СПОСОБ ПОЛУЧЕНИЯ РЕКОМБИНАНТНОГО ИНСУЛИНА ЧЕЛОВЕКА
WO2012098009A1 (fr) Polypeptide chimérique comprenant une protéine membranaire et un précurseur de l'insuline
WO2012048856A1 (fr) Proinsuline avec séquence auxiliaire
KR20000059787A (ko) 융합파트너를 이용한 재조합 단백질의 제조방법
WO2013149729A2 (fr) Proinsuline comprenant une séquence auxiliaire améliorée
RU2728611C1 (ru) РЕКОМБИНАНТНАЯ ПЛАЗМИДНАЯ ДНК pF265, КОДИРУЮЩАЯ ГИБРИДНЫЙ ПОЛИПЕПТИД, СОДЕРЖАЩИЙ ПРОИНСУЛИН ЧЕЛОВЕКА, И ШТАММ БАКТЕРИЙ Escherichia coli - ПРОДУЦЕНТ ГИБРИДНОГО ПОЛИПЕПТИДА, СОДЕРЖАЩЕГО ПРОИНСУЛИН ЧЕЛОВЕКА
RU2801248C2 (ru) Гибридный белок, содержащий фрагменты флуоресцентных белков, и его применение
CN107217069B (zh) 原核表达载体及rbFGF-2表达方法与工程菌和应用
RU2729357C1 (ru) РЕКОМБИНАНТНАЯ ПЛАЗМИДНАЯ ДНК pF267, КОДИРУЮЩАЯ ГИБРИДНЫЙ ПОЛИПЕПТИД, СОДЕРЖАЩИЙ ПРОИНСУЛИН ЛИЗПРО, И ШТАММ БАКТЕРИЙ Escherichia coli - ПРОДУЦЕНТ ГИБРИДНОГО ПОЛИПЕПТИДА, СОДЕРЖАЩЕГО ПРОИНСУЛИН ЛИЗПРО
Ponomarenko et al. Production in bacterial cell a recombinant analog of proinsulin with the proper disulfide bonds
Rani et al. Bioprocessing of inclusion bodies from E. coli. to prosduce bioactive recombinant proteins
KR20040007892A (ko) 재조합 인간 훼리틴 단백질 및 그 제조방법
Makkapati Recombinant production of peptides using SUMO as a fusion partner

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12700795

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12700795

Country of ref document: EP

Kind code of ref document: A1