Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
• • 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)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/485—Exopeptidases (3.4.11-3.4.19)
• • 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)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
  • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/14—Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
  • C12Y304/14011—Xaa-Pro dipeptidyl-peptidase (3.4.14.11)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/22—Cysteine endopeptidases (3.4.22)
  • C12Y304/22028—Picornain 3C (3.4.22.28)

Definitions

• the present invention relates to the technical fields of protein expression and protein chemistry where a matured protein is to be released from a fusion protein.
• the techniques of recombinant protein expression allow for the production of large quantities of desirable proteins which may be used for their biological activity. Such proteins are often expressed as recombinant fusion proteins in microbial host cells.
• the matured protein protein of interest
• the matured protein is often attached to a fusion partner protein or a smaller amino acid extension in order to increase the expression level, increase the solubility, promote protein folding or to facilitate the purification and downstream processing.
• Removal of the fusion partner protein from the fusion protein, to release the mature protein with native N- and C-terminus, may be pivotal for maintaining intact biological activity of the protein as well as for drug regulatory purposes.
• enterokinase which, however, lacks the specificity to be generally applicable.
• Other such enzymes are Factor Xa, trypsin, clostripain, thrombin, TEV or rhinoviral 3C protease, all of which either lacks specificity as most proteins comprise internal secondary cleavage sites or leaves an amino acid extension in the C- or N-terminal of the mature protein.
• US 8, 137,929 B2 discloses the purification of a tagged protein comprising a target protein, a purification tag and a cleavage site for in vitro cleavage by e.g. enterokinase, Factor Xa, thrombin, TEV or HRV14 3C.
• WO2008/043847 discloses a processing enzyme comprising an N-terminally attached tag derived from highly basic ribosomal proteins from thermophilic bacteria.
• WO92/10576 discloses the use of fusion proteins with DPP IV cleavable extension peptide portions in medicinal preparations.
• US 5427927 disclose a process for preparation of a desired protein by introducing an IgA protease recognition site in a precursor and cleaving the precursor with IgA protease.
• a method for release of a correctly matured protein from a fusion protein by enzymatic treatment using a picornaviral 3C protease and a Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP).
• Both picornaviral 3C protease and XaaProDAP are very specific enzymes which by the present invention are shown to exhibit complementing activities very useful for the pharmaceutical industry manufacturing protein pharmaceuticals.
• the combination of the two enzymes have the further advantage of XaaProDAP being easily removed from the matured protein by a simple gel-filtration process.
• a method for release of a matured protein from a fusion protein by enzymatic treatment using a picornaviral 3C protease and a Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP), wherein said fusion protein has the structure : Tag sequence - Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-QGP - Protein of interest, wherein
• Tag sequence is a fusion partner protein connected at its C-terminal
• Xaa1 , Xaa2, Xaa3, Xaa4 and Xaa5 are independently selected from the group consisting of the naturally occurring amino acid residues, and
• Protein of interest is a protein connected at its N-terminal and comprising said matured protein and optionally an N-terminal extension which can be cleaved by XaaProDAP or by picornaviral 3C protease and XaaProDAP.
• the XaaProDAP is able to cleave a peptide after the proline residue in a dipeptide comprising Gly-Pro or Ala-Pro.
• the picornaviral 3C protease natively has a Gly-Pro in the N- terminal. In another embodiment, the picornaviral 3C protease is HRV14 3C or a functional variant thereof.
• the XaaProDAP is an E.C. 3.4.14.1 1 enzyme or a functional variant thereof.
• the XaaProDAP is an enzyme having bacterial origin.
• the XaaProDAP is an enzyme from Lactococcus sp. or a functional variant derived from the enzyme in Lactococcus sp.
• a fusion enzyme comprising the catalytic domains of a picornaviral 3C protease and a XaaProDAP or functional variants thereof.
• the fusion enzyme comprises the full length of a picornaviral 3C protease and the full length XaaProDAP or functional variant thereof.
• the fusion enzyme comprises a combination of either a catalytic domain or full-length picornaviral 3C protease with either a catalytic domain or full-length XaaProDAP or functional variants thereof.
• Figure 4 Mass spectrum of Reaction 3 where RL27_HRV14_PYY(3-36) is incubated with XaaProDAP and HRV14 protease for 1 hour at 37 °C using a 1 :1 :20 molar ratio.
• X-axis Mass over charge ratio (m/z) in Da.
• Y-axis Relative intensity.
• Figure 7 Mass spectrum of Reaction 6 where RL27_HRV14_PYY(3-36) is incubated with XaaProDAP and HRV14 protease for 3 hours at 37 °C using a 1 :1 :10 molar ratio.
• X-axis Mass over charge ratio (m/z) in Da.
• Y-axis Relative intensity.
• a method for release of a matured protein from a fusion protein by enzymatic treatment using a picornaviral 3C protease and a Xaa-Pro-dipeptidyl aminopeptidase (XaaProDAP).
• the method of the invention provides a number of advantages over previously described methods for release of a matured protein from a fusion protein. For example, it has been surprisingly found that a very specific hydrolysis of the fusion protein can be obtained such that the mature protein is released with the correct native N-terminal amino acid in the absence or with a minimum level of related impurities and in high yields. The presence of any related impurities, i.e. proteins resembling the mature protein by having limited differences in chemical structure, is clearly undesirable as they are difficult and thus expensive to remove in a manufacturing process.
• Certain embodiments of the invention have the further advantage of allowing release of the matured protein from the fusion protein in a single process step. Additional embodiments have the advantage of allowing release of the matured protein from the fusion protein at reactions conditions having low temperatures.
• Xaa-Pro dipeptidyl aminopeptidase (“XaaProDAP”) as used herein is intended to mean an enzyme having dipeptidase activity specific for Xaa-Pro dipeptides, i.e. the scissile bond connecting the C-terminal of the Xaa-Pro dipeptide with the N-terminal of a peptide or protein.
• XaaProDAP ' s are classified according to the international union of Biochemistry and molecular Biology Enzyme (IUBMB) Enzyme Nomenclature as the enzymes EC 3.4.14.1 1 from the peptidase family S15 and as the enzymes EC 3.4.14.5 from the peptidase family S9B.
• Non-limiting examples of XaaProDAP are dipeptidyl-peptidase IV (DPP-IV) from mammals.
• Other non-limiting examples of XaaProDAP are Xaa-Prolyl dipeptidyl
• Xaa-Prolyl dipeptidyl aminopeptidase from Streptococcus suis has the sequence : MRFNQFSFIKKETSVYLQELDTLGFQLIPDASSKTNLETFVRKCHFLTANTDFALSNMIAEWD TDLLTFFQSDRELTDQIFYQVAFQLLGFVPGMDYTDVMDFVEKSNFPIVYGDIIDNLYQLLNT RTKSGNTLIDQLVSDDLIPEDNHYHFFNGKSMATFSTKNLIREWYVETPVDTAGTGQTDIVK LSILRPHFDGKIPAVITNSPYHQGVNDVASDKALHKMEGELAEKQVGTIQVKQASITKLDLDQ RNLPVSPATEKLGHITSYSLNDYFLARGFASLHVSGVGTLGSTGYMTSGDYQQVEGYKAVI DWLNGRTKAYTDHTRSLEVKADWANGKVATTGLSYLGTMSNALATTGVDGLEVIIAEAGISS WYDYYRENGLVTSPGG
• a non-limiting example of a functional variant is an analogue, an extended or a truncated version of a naturally occurring XaaProDAP which functional variant retain dipeptidase activity specific for Xaa-Pro dipeptides.
• the 3C proteases are a group of cysteine proteases with a serine proteinase-like fold that are responsible for generating mature viral proteins from a precursor polyprotein in vira from the Picornaviridae family.
• Picornaviral 3C protease as used herein is intended to mean a protease originating from the Picorna viridae including functional variants thereof, which protease cleave the peptide bond between a P1 -P1 ' Gln-Gly pair where the scissile bond connects Gin and Gly (where P1 and P1 ' according to commonly used notation denote the first amino acids on the N- terminal and C-terminal sides of the scissile bond, respectively).
• P1 and P1 ' according to commonly used notation denote the first amino acids on the N- terminal and C-terminal sides of the scissile bond, respectively.
• P2' denote the second amino acid on the C-terminal side of the scissile bond.
• Enzymes with this substrate specificity are typically isolated from virus of the genus enterovirus, which currently comprises
• Non-limiting examples of such picornian 3C proteases are Human Rhino Virus 14 3C (HRV14 3C) protease having the sequence
• the picornaviral 3C protease may be an enzyme naturally occurring in the Picorna viridae, but it may also be a functional variant of such an enzyme.
• a non-limiting example of a functional variant is an analogue, an extended or a truncated version of a naturally occurring picornaviral 3C protease which functional variant retain substrate specificity for the Gln-Gly pair.
• Picornaviral 3C protease is commercially available as e.g. His-tagged HRV14 3C (Sino Biological Inc), GST-tagged HRV14 3C (Speed Biosystems) and as dual-tagged (GST plus His) HRV14 3C (Thermo Scientific Pierce).
• the picornaviral 3C protease cleaves the peptide bond between a P1-PV Gin-Ala pair where the scissile bond connects Gin and Ala. In another embodiment the picornaviral 3C protease cleaves the peptide bond between a P1 -PV GIn-Ser pair where the scissile bond connects Gin and Ser. In another embodiment the picornaviral 3C protease natively has a proline as the second amino acid residue from the N-terminal, or the picornaviral 3C protease is an analog of a picornaviral 3C protease which natively has a proline as the amino acid residue in position 2 from the N-terminal. In another embodiment the picornaviral 3C protease natively has a Gly-Pro in the N-terminal.
• “Matured protein” as used herein is intended to mean a protein or a polypeptides of interest, or an extended version thereof which extended version can be cleaved at its N- terminus by XaaProDAP.
• the matured protein is often present as a fusion protein during its manufacture, such as a protein comprising a tag sequence, an optional linker sequence, a picornaviral 3C protease site in addition to the matured protein.
• Non-limiting examples of a mature protein is glucagon, PYY, GLP-1 (7-37), Arg34-GLP1 (7-37), Arg34-GLP-1 (9-37) and Arg34-GLP-1 (1 1 -37).
• Arg34-GLP-1 (7-37) is K34R-GLP-1 (7-37).
• Fusion protein as used herein is intended to mean a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences encoding at least two proteins.
• a fusion protein can comprise a tag protein fused with a protein having an activity of pharmaceutical interest. Fusion proteins are often used for improving recombinant expression of therapeutic proteins as well as for improved recovery and purification of such proteins from cell cultures and the like. Fusion proteins may also comprise synthetic sequences, e.g. a linker sequence.
• Fusion partner protein as used herein is intended to mean a protein which is part of a fusion protein, i.e. one of the at least two proteins encompassed by the fusion protein.
• fusion partner proteins are tag proteins and solubilisation domains such as His6-tags, Maltose-binding protein, Thioredoxin, etc.
• Fusion enzyme as used herein is intended to mean a fusion protein comprising at least two proteins which are both enzymes (in the sense that the two proteins have backbone sequences that are covalently connected).
• Tag protein as used herein is intended to mean a protein which is attached to another protein in order to facilitate or improve the manufacture of said other protein, e.g. facilitating or improving the recombinant expression, recovery and/or purification of said other protein.
• Non-limiting examples of tag proteins are His6-tags, Glutathione S-transferase (GST), Maltose-binding Protein (MBP), Staphylococcus aureus protein A, biotinylated peptides and highly basic proteins from thermophilic bacteria as described in
• Tag sequence as used herein is intended to mean a sequence comprising a protein.
• a tag sequence may optionally also comprise an additional sequence, e.g. a linker sequence.
• Protein tags are peptide sequences genetically grafted onto a recombinant protein,which may be removable by chemical agents or by enzymatic means, such as proteolysis. Tags are attached to proteins for various purposes
• Linker as used herein is intended to mean an amino acid sequence which is typically used to facilitate the function, folding or expression of fusion proteins. It is known in the art that two proteins present in the form of a fusion enzyme may interact with the enzyme activities of each other, an interaction that can often be eliminated or reduced by the insertion of a linker between the two enzyme sequences.
• analogues as used herein is intended to mean proteins which are derived from another protein by means of substitution, deletion and/or addition of one or more amino acid residues from the protein.
• analogues of GLP-1 (7-37) are K34R-GLP- 1 (7-37) where residue 34 has been substituted by an arginine residue and K34R-GLP-1 (9- 37) where residue 34 has been substituted with an arginine residue and amino acid residues 7-8 have been deleted (using the common numbering of amino acid residues for GLP-1 peptides).
• “Functional variant” as used herein is intended to mean a chemical variant of a certain protein which has an altered sequence of amino acids but retains substantially the same function as the original protein.
• functional variants are e.g. extended proteins, truncated proteins, fusion proteins and analogues.
• Non-limiting examples of functional variants of HRV14 3C are e.g. His6 tagged HRV14 3C and GST-tagged HRV14 3C.
• Non-limiting functional variants of GLP-1 (7-37) are K34R-GLP-1 (7-37).
• a function variant of a protein comprises from 1 -2 amino acid substitutions, deletions or additions as compared said protein. In another embodiment, a functional variant comprises from 1-5 amino acid substitutions, deletions or additions as compared to said protein. In another embodiment, a functional variant comprises from 1 -15 amino acid substitutions, deletions or additions relative to the corresponding naturally occurring protein or naturally occurring sub-sequence of a protein. In yet another
• a functional variant of a protein analogue in the overlapping sequence comprises no more than 10 amino acids different from the sequence of the native protein. .
• a functional variant of a protein analogue in the overlapping sequence comprises no more than 5 amino acids different from the sequence of the native protein.
• solubilisation domain as used herein is intended to mean a protein which is part of a fusion protein and which is to render said fusion protein more soluble than the fusion partner protein itself under certain conditions.
• solubilisation domains are DsbC (Thiohdisulfide interchange protein), RL9 (Ribosomal Protein L9) as described in WO2008/043847, MBP (Maltose-binding Protein), NusA (Transcription termination/antitermination protein) and Trx(Thioredoxin).
• enzyme treatment is intended to mean a contacting of a substrate protein with an enzyme which catalyses at least one reaction involving said substrate protein.
• One common enzymatic treatment is the contacting of a fusion protein with an enzyme having proteolytic activity in order to separate two proteins being constituents of the fusion protein.
• PBS Phosphate buffered saline
• a typical PBS buffer used for enzymatic reactions in the present invention is (8.05 mM Na2HP04x2H20, 1 ,96 mM KH2P04, 140 mM NaCI, pH 7.4).
• Tag sequence is a fusion partner protein connected at its C-terminal
• Xaa1 , Xaa2, Xaa3, Xaa4 and Xaa5 are independently selected from the group consisting of the naturally occurring amino acid residues, and
• Protein of interest is a protein connected at its N-terminal and comprising said matured protein and optionally an N-terminal extension which can be cleaved by XaaProDAP or by picornaviral 3C protease and XaaProDAP.
• the protein of interest (or the matured protein) is GLP-1 , Glucagon, PYY , amylin or a functional variant thereof.
• Xaa2-Xaa3-Xaa4-Xaa5 is EVLF.
• Xaa1 is selected from L and S.
• Xaa1 -Xaa2-Xaa3-Xaa4-Xaa5 is selected from LETLF (SEQ ID NO:16) and LFAQT (SEQ ID NO:17).
• the method of the present invention form a very low amount of product related impurities for most proteins.
• particularly low amounts of product related impurities may arise when a) the amino acid residue at position 2 from the N-terminal of said matured protein is different from proline, b) the amino acid residue at position 2 from the N-terminal of said matured protein is different from alanine, and/or 3) the N-terminal amino acid residue of said matured protein is different from proline.
• said enzymatic treatment is done after the fusion protein has been isolated from an expression host.
• the enzymatic treatment may be done by simultaneously contacting said fusion protein with said picornaviral 3C protease and said XaaProDAP.
• the enzymatic treatment may be done by first contacting said fusion protein with said picornaviral 3C protease to separate a tag from the fusion protein, and then contacting said de-tagged protein with said XaaProDAP to form said matured protein.
• said picornaviral 3C protease may be inactivated or separated from the de- tagged protein prior to contacting said de-tagged protein with said XaaProDAP to form said matured protein.
• the XaaProDAP may be added to a reaction mixture containing the picornaviral 3C protease, fusion protein and de-tagged protein once the majority of the fusion protein has been cleaved by picornaviral 3C protease into the de- tagged protein.
• the enzymatic treatment to liberate the matured protein may be done at a pH in the range from about 6 to about 9 , such as in the range from about 7 to about 8.5, in the range from about 7.5 to about 8.5, in the range from about 8 to about 9, or in the range from about 6 to about 7.
• Suitable temperatures for the enzymatic treatment are in the range from about 0 °C to about 50 °C, in the range from 30 °C or 37 °C, in the range from about 0 °C to about 15 °C, in the range from about 0 °C to about 10 °C, in the range from about 2 °C to about 10 °C, in the range from about 5 °C to about 15 °C, in the range from about 0 °C to about 5 °C, or in the range from about 2 °C to about 8 °C.
• the enxymatic treatment is done at a pH in the range from about pH 7.5 to about pH 8.5 and at a temperature in the range from about 4 °C to about 10 °C.
• Suitable reaction times for the enzymatic treatment are in the range for about 1 minute to about 3 hours, for about 3 hours to about 24 hours, for about 3 hours to about 16 hours, for about 6 hours to about 24 hours, or for about 10 hours to about 16 hours.
• Suitable buffers for the enzymatic treatment are various buffers exhibiting buffer capacity at the desired pH for the enzymatic treatment.
• the enzymatic treatment is done in the presence of a buffer selected from PBS, TRIS and HEPES.
• the picornaviral 3C protease and thed XaaProDAP are co-expressed in an expression host.
• the picornaviral 3C protease and the XaaProDAP are expressed as a fusion enzyme in an expression host.
• the fusion protein is co-expressed in an expression host together with the picornaviral 3C protease and the XaaProDAP.
• the picornaviral 3C protease, the XaaProDAP as well as the fusion protease may be produced by means of recombinant nucleic acid techniques.
• a cloned wild- type picornaviral 3C protease or XaaProDAP nucleic acid sequence is modified to encode the desired protein. This modification can also include the in-frame fusion of the nucleic acid sequences encoding two or more proteins to be expressed as a fusion protein.
• Such a fusion protein can be the fusion protease (picornaviral 3C protease and XaaProDAP) as well as the fusion protease, picornaviral 3C protease or XaaProDAP fused to e.g. a His-tag or a solubilization domain (DsbC, RL9, MBP, NusA or Trx).
• This modified sequence is then inserted into an expression vector, which is in turn transformed or transfected into expression host cells.
• the nucleic acid construct encoding the picornaviral 3C protease, XaaProDAP or fusion protease may suitably be of genomic, cDNA or synthetic origin. Amino acid sequence alterations are accomplished by modification of the genetic code by well known techniques.
• a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
• the vector is preferably an expression vector in which the DNA sequence encoding the picornaviral 3C protease, XaaProDAP or fusion protease is operably linked to additional segments required for transcription of the DNA.
• operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide until it terminates within a terminator.
• expression vectors for use in expressing picornaviral 3C protease are provided.
• XaaProDAP or fusion protease will comprise a promoter capable of initiating and directing the transcription of a cloned gene or cDNA.
• the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
• expression vectors for use of expression of picornaviral 3C protease, XaaProDAP or fusion protease will also comprise a terminator sequence, a sequence recognized by a host cell to terminate transcription.
• the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
• picornaviral 3C protease, XaaProDAP or fusion protease can be aimed for either intracellular expression in the cytosol of the host cell or be directed into the secretory pathway for extracellular expression into the growth medium.
• Intracellular expression is the default pathway and requires an expression vector with a DNA sequence comprising a promoter followed by the DNA sequence encoding the picornaviral 3C protease, XaaProDAP or fusion protease polypeptide followed by a terminator.
• a secretory signal sequence (also known as signal peptide or a pre sequence) is needed as an N-terminal extension of the picornaviral 3C protease, XaaProDAP or fusion protease.
• a DNA sequence encoding the signal peptide is joined to the 5' end of the DNA sequence encoding the picornaviral 3C protease, XaaProDAP or fusion protease in the correct reading frame.
• the signal peptide may be that normally associated with the protein or may be from a gene encoding another secreted protein.
• XaaProDAP or fusion protease is introduced may be any cell that is capable of expressing picornaviral 3C protease, XaaProDAP or fusion protease either intracellular ⁇ or
• suitable host cells include yeast, fungi, insects and higher eukaryotic cells such as mammalian cells.
• the picornaviral 3C protease and the XaaProDAP may be co-expressed in an expression host.
• the picornaviral 3C protease and the XaaProDAP may be expressed as a fusion enzyme in an expression host.
• said fusion protein is co-expressed in an expression host together with said picornaviral 3C protease and said XaaProDAP, thus allowing the manufacture of the 3C protease and the XaaProDAP simulataneously with the enzymatic treatment of the fusion protein to liberate the matured protein.
• suitable promoters for directing the transcription of the nucleic acid constructs in a bacterial host cell are, for expression in E. coli, the promoters obtained from the lac operon, the trp operon and hybrids thereof trc and tac, all from E. coli (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21 -25).
• Other even stronger promoters for use in E. coli are the bacteriophage promoters from T7 and T5 phages.
• the T7 promoter requires the presence of the T7 polymerase in the E. coli host (Studier and Moffatt, J. Mol. Biol. 189, 1 13, (1986)).
• E. coli also has strong promoters for continuous expression, eg. the synthetic promoter used to express hGH in Dalb0ge et al, 1987, Biotechnology 5, 161-164.
• Bacillus subtilis levansucrase gene Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes are suitable examples. Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.
• Effective signal peptide coding regions for bacterial host cells are, for E. coli, the signal peptides obtained from the genes DegP, OmpA, OmpF, OmpT, PhoA and Enterotoxin STII, all from E. coli.
• signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137. For both E. coli and Bacillus, signal peptides can be created de novo according to the rules outlined in the algorithm SignalP (Nielsen et al, 1997, Protein Eng. 10, 1 -6., Emanuelsen et al, 2007, Nature Protocols 2, 953-971 ). The signal sequences are adapted to the given context and checked for SignalP score.
• Examples of preferred expression hosts are E. coli K12 W31 10, E. coli K12 with a trace of B, MC1061 and E. coli B BL21 DE3, harbouring the T7 polymerase by lysogenization with bacteriophage ⁇ . These hosts are selectable with antibiotics when transformed with plasmids for expression.
• the preferred host is eg. E. coli B BL21 DE3 3xKO with deletion of the 2 D,L-alanine racemase genes Aalr, AdadX, and deletion of the Group II capsular gene cluster ⁇ (kpsM-kpsF), specific for E. coli B and often associated with pathogenic behaviour.
• the deletion of the Group II gene cluster brings E. coli B BL21 DE3 3xKO into the same safety category as E. coli K12. Selection is based on non- requirement of D-alanine provided by the air gene inserted in the expression plasmid instead of the AmpR gene.
• Fusion protein may be done in a similar way by bacterial expression as described above for the picornaviral 3C protease, XaaProDAP or fusion protease.
• picornaviral 3C protease, XaaProDAP or a fusion protease may be recoved and purified to the required purity by conventional techniques.
• conventional recovery and purification techniques are centrifugation, solubilization, filtration, precipitation, ion-exchange chromatography, immobilized metal affinity chromatography (IMAC), RP-HPLC, gel-filtration and freeze drying.
• picornaviral 3C protease examples include Cordingley et al., J. Virol. 1989, 63, pp5037-5045, Birch et al., Protein Expr Purif., 1995, 6, pp609-618 and WO2008/043847.
• picornaviral 3C protease originates from a virus selected from Enterovirus, Coxsackievirus, Cowpea mosaic comovirus, Rhinovirus and Poliovirus.
• analogue in the overlapping sequence comprises no more than 10 amino acids different from the sequence of the native protein.
• Tag sequence is a fusion partner protein connected at its C-terminal
• Xaa1 , Xaa2, Xaa3, Xaa4 and Xaa5 are independently selected from the group consisting of the naturally occurring amino acid residues, and
• Protein of interest is a protein connected at its N-terminal and comprising said matured protein and optionally an N-terminal extension which can be cleaved by XaaProDAP or by picornaviral 3C protease and XaaProDAP.
• Xaa1 -Xaa2-Xaa3-Xaa4- Xaa5 is selected from LETLF (SEQ ID NO: 16) and LFAQT (SEQ ID NO: 17).
• treatment using a picornaviral 3C protease and a XaaProDAP is done by using a fusion enzyme comprising a picornaviral 3C protease and a XaaProDAP.
• Fusion enzyme comprising the catalytic domains of a picornaviral 3C protease and a XaaProDAP.
• fusion enzyme according to any of embodiments 52-56 comprising an amino acid residue linker between said picornaviral 3C protease or the catalytic domain thereof and said XaaProDAP or the catalytic domain thereof, where said linker has a length of from 2 to 100 amino acid residues.
• the pET system was used for expression of enzymes as this system provides a powerful means of expressing proteins in E.coli.
• target genes are cloned under control of strong bacteriophage T7 transcription and translation signals, and expression is induced by providing a source of T7 RNA polymerase in the host cell.
• E.coli expression plasmids (pET22b, Novagen) encoding Xaa-Prolyl Dipeptidyl aminopeptidase (XaaProDAP, full length) from Lactococcus lactis subsp. cremoris CNCM I- 1631 (SEQ ID NO:1 ) with different N-terminal extensions/fusion partners were prepared.
• One plasmid comprised a His6 tag in the N-terminal with the sequence MHHHHHH
• the gene fragments encoding full-length of XaaProDAP (SEQ ID NO:1 ), His6 tags, fusion partners and linkers were codon-optimized for expression in E.coli and prepared by gene synthesis (GenScript).
• GGSSGSGSELRTQSGS (SEQ ID NO:9) was introduced adjacent to the N-terminal amino acid of the XaaProDAP sequence to allow enzymatic separation of the fusion partner from the XaaProDAP enzyme if needed.
• This linker region also contained an Xhol site and BamHI site, which allows introduction of another N-terminal extension to the enzyme.
• the XaaProDAP sequence was prepared with small 3'-end overhang encoding GSS, which includes a Sacl restriction site that allows introduction C-terminal extensions to the XaaProDAP enzyme.
• the plasmid constructs specified in Table 1 were generated by DNA synthesis and subcloning into pET22b vectors using standard cloning technologies known to those of ordinary skill in the art (obtained from GenScript):
• Expression plasmids were transformed into E.coli BL21 (DE3) and expressed in small scale.
• E.coli BL21 (DE3) were transformed with plasmid using a procedure based on Heat Shock at 42°C according to the manufacturer. Transformed cells were plated onto LB agar plates and incubate overnight at 37°C with 10 mg/L ampicillin. Overnight Terrific broth (TB) culture with 0.5% glucose and 50 mg/L carbenicillin of each transformant was prepared at 30°C and shaking at 700 rpm using a Glas-Col shaker (Glas-Col). 20 ⁇ of overnight culture of each transformant was used to inoculate 0.95 ⁇ of TB medium with 50 mg/L carbenicillin in 96 Deep-Well plates (2 ml) and transformants were propagated overnight at 700 rpm.
• TB Terrific broth
• Purification screen Small scale purification using IMAC resin was performed to evaluate the combined expression and purification potential of each construct.
• 250 ⁇ _ of lysis buffer 50 mM NaP04, 300 mM NaCI, 10 mM Imidiazole, 10mg/ml Lysozyme, 2501 ⁇ / ⁇ _ Benzoase and 10% DDM
• 250 ⁇ _ of lysis buffer 50 mM NaP04, 300 mM NaCI, 10 mM Imidiazole, 10mg/ml Lysozyme, 2501 ⁇ / ⁇ _ Benzoase and 10% DDM
• Debris was removed by centrifugation, the supernatant was filtered (0.45 ⁇ ) and transferred onto 1.2 ⁇ filter plates containing Ni2+-loaded Sepharose Fast Flow (prepared from washing 30 ⁇ _ of a 50% slurry in 20% EtOH) (GE Healthcare).
• the supernatant was incubated for 20 min by shaking at 400 rpm with resin to bind the protein and the solute was removed by gentle centrifugation at 100xg for 1 min.
• the resin was washed with 50mM sodium phosphate, 300 mM NaCI, 30 mM Imidiazole, pH 7.5 by gentle mixing and the resin was dried by centrifugation.
• 40 ⁇ _ of elution buffer 50 mM sodium phosphate, 300 mM NaCI, 300 mM Imidazole
• XaaProDAP all constructs gave rise to clear protein bands of the expected size following Coomasie Blue staining of the gels.
• the eluted protein was also evaluated using SDS-PAGE.
• His6, RL9, Trx, DsbC and NusA tagged variants of XaaProDAP gave rise to a clear protein band of the expected size following Coomassie staining of gels, showing that the L. lactis XaaProDAP enzyme could be expressed as soluble protein with a range of different fusion partners and captured using IMAC.
• BL21 (DE3) transformants (from a glycerol stock) harbouring the pET22b plasmid encoding His-Trx-XaaProDAP was propagated overnight in 50 ml of Terrific Broth medium containing 50 mg/L Carbenicillin and 0.5% glucose by shaking at 37°C with 100 rpm
• BufferA 50 mM sodium phosphate, 300 mM NaCI, 10 mM imidazole pH 7.5
• Buffer B 50 mM sodium phosphate, 300 mM NaCI, 300 mM imidazole pH 7.5
• Buffer C 50 mM sodium phosphate, 300 mM NaCI, 30 mM imidazole pH 7.5
• the column was initially equilibrated for 10 column volumes of buffer A. After loading of the application, unbound protein was removed by washing using 7 column volumes of buffer C. A step elution from 0-100 % buffer B for 5 column volumes was used to elute the XaaProDAP protease from the column.
• SDS-PAGE was used to analyse the recoved fractions and showed a clear band of the expected size corresponding to His-Trx-XaaProDAP.
• the purest fractions were pooled and loaded onto a HiLoad S200 16/600 (GE-Healthcare) gel filtration column. And size separation was performed with a flow rate of 1 .2 ml/min using 1X PBS buffer (phosphate buffered saline, pH 7.4). Collected fraction of the predominant peak were analysed by SDS- PAGE and a clear band of the expected size was observed. The purity was estimated to be at least 90% as judged by SDS-PAGE and HPLC analysis.
• Plasmid constructs and expression of model fusion proteins containing basic tag Plasmid constructs and expression of model fusion proteins containing basic tag.
• a basic tag comprising Ribosomal Protein L27 from T. maritima, previously described in WO2008/043847 was used as a fusion partner and has the sequence MAHKKSGGVAKNGRDSLPKYLGVKVGDGQIVKAGNILVRQRGTRFYPGKNVGMGRDFTLF ALKDGRVKFETKNNKKYVSVYEE (SEQ ID NO: 10).
• the fusion proteins were designed so that the RL27 fusion partner can be removed by HRV14 3C enzyme and the remaining GP sequence can be removed by XaaProDAP.
• a flexible linker containing a HRV14 cleavage site was used to link the basic tag to the model peptide sequences and had the sequence SSSGGSEVLFQGP (SEQ ID NO: 1 1 ).
• the model peptide sequences used were human pancreatic peptide YY (PYY(3-36)), Glucagon and Glucagon-like peptide-1 (7-37,K34R) (GLP-1 (7-37,K34R)) having the following sequences:
• Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 13)
• E.coli expression plasmids (pET22b, Novagen) were prepared such that they encoded the three fusion proteins as specified in Table 2. Table 2. Fusion proteins encoded by plasmid constructs using pET22b vectors.
• Gene fragments codon-optimized for E.coli and spanning the entire fusion proteins were made by gene synthesis and ligated into the cloning site of the pET22b vector using standard cloning techniques (obtained from GenScript).
• RL27_HRV14_GLP-1 (7-37,K34R) was done as follows: E.coli BL21 (DE3) was transformed with the plasmid and plated on LB agar plates containing 100 mg/L ampicillin and overnight cultures were dissolved in 10 ml LB medium and used to inoculate 750 ml LB with 50mg/ml Carbenicillin in shaker flasks. Shaker flasks were incubated at 100 rpm at 37°C. When OD600 of 0.4 was reached protein expression was induced by adding 0.3 mM IPTG and cells were harvested by centrifugation following 3 hours incubation at 37°C.
• Buffer A 50mM sodium phosphate, pH 7.0
• Buffer B 50mM sodium phosphate, 1000mM NaCI, pH 7.0
• the fusion proteins were eluted from the columns using Buffer B.
• the proteins were purified by gel filtration essentially as described in Example 2, but using a S75 16/600 column (GE-Healthcare) for the separation.
• the purified proteins were evaluated by SDS- PAGE analysis and the correct intact mass was verified by LC-MS. UV280 was used to determine the concentration of the fusion proteins.
• the purified His-Trx-LLXaaProDAP was prepared as described in Example 2.
• a purified variant of the HRV14 protease was used for the experiments, which had an N- terminal basic tag (ribosomal L9 from T. maritima described previously in WO2008/043847).
• This protease was named RL9-HRV14 3C and was prepared essentially as described previously, using a SP FF for capture and gel filtration for polishing.
• the RL9-HRV14 3C protease was identical to the one described in WO2008/043847 and had the following sequence:
• Solvent A 0.1 % formic acid in H20
• Solvent B 99,9% MeCN, 0.1 % formic acid(v/v) Liquid Chromatography was performed with the following gradient to separate the enzyme digests
• the recorded mass spectra were deconvoluted and analysed using the Bruker Compass dataanalysis version 4.1 software (Bruker Daltonics) with adjusted settings for mass ranges (2000-17000 Da) and resolutions (>20.000) according to manufactorers instructions.
• the UV215 nm chromatogram and total ion count (TIC) chromatograms were evaluated in parallel, to ensure that there was agreement between MS data obtained and UV215 nm traces of the peptides.
• the experimental determined masses indicated in the following examples refers to the most abundant mass, e.g. the mass of the molecule with the most highly represented isotope distribution, based on the natural abundance of the isotopes of the protein detected. In the following, the mass spectrometry data was obtained with a mass accuracy lower than 100 ppm.
• Enzymatic reactions with RL27_HRV14_PYY(3-36) as a fusion protein model substrate The concentration RL27-HRV14-PYY(3-36) was adjusted to a concentration of 1 mg/ml. Enzymatic reaction were setup in reaction volumes of 100 ⁇ using 1 x PBS, pH 7.4 as enzyme reaction buffer.
• His-Trx-LLXaaProDAP RL9-HRV14 3C: RL27-HRV14-PYY(3-36) substrate was setup in molar ratios of 1 :1 :40 or 1 :1 :20, respectively, and the reactions were carried out at 37°C for 1 or 3 hours (as depicted in Table 3): The enzymatic reactions were stopped by addition >0.5 M AcOH prior to LC-MS analysis Table 3: Enzymatic reactions using co-treatment of His-Trx-LLXaaProDAP and RL9-HRV14 3C with RL27_HRV14_PYY(3-36) from Example 3. Experimentally determined predominant peaks detected in deconvoluted mass spectra of reactions 1 -6 are indicated.
• Peak 3# was observed with -30 % of the relative intensity of Peak#1 and corresponded to GP-PYY(3-36) and Peak 4# corresponded to the intact RL27-HRV14- PYY(3-36) fusion protein (Fig. 2). This indicates, incomplete cleavage with both His-Trx- XaaProDAP and the RL9-HRV14 3C protease following one hour incubation at these reaction conditions. Reaction 2 shows that 3 hours of incubation completely removes the full length protein and leaves the released mature PYY(3-36) and the RL27 tag (Fig. 3). The GP- PYY(3-36) peak was still present but with reduced intensity (Fig. 3).
• the data demonstrates that the combined action of XaaProDAP and HRV14 activity can specifically release a fully maturated PYY(3-36) peptide with the correct N-terminal amino acid from a fusion protein and proofs the value of using these two enzymes for processing of fusion proteins.
• the concentrations of model fusion proteins was adjusted to a concentration of 0.5 mg/ml (in PBS buffer pH 7.4).
• Three enzymatic reaction were setup in reaction volumes of 55 micro litre using PBS, pH 7.4 as reaction buffer.
• His-Trx-LLXaaProDAP: RL9-HRV14 3C: RL27_HRV14_Glucagon was setup in molar ratios of either 1 :100:2000 or 1 :500:10000 as depicted in Table 4.
• the enzymatic reactions were carried out for 3 hours at 37°C and stopped by addition of >0.5 M AcOH and LC-MS analysis was conducted as described in Example 4.
• the concentrations of model fusion proteins was adjusted to a concentration of 0.5 mg/ml (in PBS buffer pH 7.4).
• the following enzymatic reaction were setup in reaction volumes of 55 micro litre using PBS, pH 7.4 as reaction buffer.
• Molar ratios which were evaluated was either 1 :100:2000 or 1 :500:10000 of His-Trx- LLXaaProDAP: RL9-HRV14 3C: GLP-1 (7-37, K34R) as depicted in Table 5.
• the enzymatic reactions were carried out for 3 hours at 37°C and stopped by addition of >0.5 M AcOH and LC-MS analysis was conducted as described in Example 4.
• reaction 10 For reaction 10 an additional predominant peak with a mass of 3537.76 Da (Peak #3) and an intensity approximately equal to GLP-1 (7-37, K34R) peak#1 was also clearly detected and this mass corresponded to a GP extended version of GLP-1 (7-37, K34R). This indicates that the XaaProDAP amount used in Reaction 10 is not sufficient to obtain full processing. However, as the 3537.76 Da peak was not observed in reaction 9, the our data proofs that a reaction using combined actions of HRV14 and XaaProDAP can be optimized to deliver correctly processed and maturated GLP-1 analogs with the correct N-terminal amino acid and with a minimum amount of unwanted cleavage products.
• picornaviral 3C proteases are active at low temperature, which is beneficial for industrial process. It was therefore evaluated whether XaaProDAP in combination with HRV14 could work at lower temperatures.
• concentrations of model fusion proteins were adjusted to 0.5 mg/ml (in PBS buffer pH 7.4).
• the following enzymatic reaction was setup in reaction volumes of 30 micro litre using PBS, pH 7.4 as reaction buffer.
• His-Trx-LLXaaProDAP RL9-HRV14:RL27_HRV14:GLP-1 (7-37,K34R)

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