WO2016034534A1 - Pré-séquences basiques riches en lysine - Google Patents

Pré-séquences basiques riches en lysine Download PDF

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WO2016034534A1
WO2016034534A1 PCT/EP2015/069846 EP2015069846W WO2016034534A1 WO 2016034534 A1 WO2016034534 A1 WO 2016034534A1 EP 2015069846 W EP2015069846 W EP 2015069846W WO 2016034534 A1 WO2016034534 A1 WO 2016034534A1
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protein
sequence
amino acid
recombinant fusion
fusion protein
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PCT/EP2015/069846
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English (en)
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Christine Bruun SCHIØDT
Anja Kallesøe PEDERSEN
Jianhe Chen
Zhiru Yang
Wenjuan XIA
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Novo Nordisk Health Care Ag
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Publication of WO2016034534A1 publication Critical patent/WO2016034534A1/fr

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    • 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)
    • C12Y304/14001—Dipeptidyl-peptidase I (3.4.14.1), i.e. cathepsin-C
    • 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
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention relates to the field of recombinant protein expression and purification.
  • Protein purification tags are widely used to increase the ease of the purification of recombinant proteins. In order to obtain a wild type protein, it is advantageous to use a purification tag that can easily be removed.
  • the amino acids sequence of the pre-sequence or tag may influence both expression level and enzymatic processing by endogenous enzymes in E. coli as well as any subsequent processing or modifications.
  • methionine amino peptidase (cleaves off the N-terminal Met residue, provided the penultimate residue is small, e.g. Ser, Pro, Gly, Ala, Thr, Val)(Hirel et al (1989) PNAS 86, 8247-8251 , Giglione et al (2004) Cell. Mol. Life 61 , 1455-1474) and c) methyltransferase capable of methylating N- terminal Met residues.
  • dipeptidyl peptidase I (DPPI) (Yang et al (201 1 ) Protein Expr. Purif. 76, 59-64). Broad applicability of dipeptidyl peptidase has previously been demonstrated by cleavage of a dipeptide-p-nitroanilide substrate and N-terminally extended MEAE-hGH (Met-Glu-Ala-Glu-human growth hormone)(Yang et al (201 1 ) Protein Expr. Purif. 76, 59-64)
  • the specificity of the DPPI enzyme requires a free primary amine in the N-terminal and that the di-peptides (Xaa-Xbb) do not contain any Pro or Cysteine residues or a positively charged residue in the Xaa position.
  • a plurality of combinations of dipeptide can be processed by DPP1 (Yang et al (201 1 ) Protein Expr. Purif. 76, 59-64, McDonald et al (1969) J.Biol.Chem. 244, 2693-2709 and McGuire et al (1992) Arch. Biochem. Biophys 295, 280-288).
  • WO07025987 describes expression of tagged hlL-21 molecules and subsequent cleavage of
  • WO08104513 describes further tagged hll-21 molecules where H is substituted by M, S or T.
  • a general problem for heterologous protein expressing in E. coli is the removal of host cell proteins. This is in particular true for proteins having a pi value close to 5 as a large proportion of the E. coli proteasome have pi values around 5 (Tonella et al, (1998) Electrophor. 19, 1960-1971 ). Summary
  • the present invention pertains to a group of pre-sequences or tags that improves expression, processing and/or purification of a heterologous protein when expressed in a suitable host strain such as E. coli.
  • N-terminal pre-sequence has been optimised to enable high expression levels, a highly efficient method for purification and removal of the pre-sequence by a protease to produce native hGH protein.
  • the present invention relates to the finding that the production process for producing a recombinant protein can be improved by applying a novel pre-sequence or protein tag.
  • An extended tag including at least three lysines has spectacular advantages as is described herein below.
  • DPPI dipeptidyl peptidase I
  • the pi of the fusion protein may increase around 1 pH unit.
  • the method of the present invention takes advantage of this pi change by including a precipitation step at low pH, selected to keep the recombinant fusion protein in soluble form while a substantial amount of the host cell proteins precipitates at this low pH.
  • a simple clarification step a large fraction of host cell proteins can be removed before further purification of the recombinant fusion protein.
  • the protein may subsequently be purified by any standard purification step such as by chromatography.
  • the initial clearance also has a positive effect on downstream purification steps as binding capacity and efficiency of a subsequent capture column are greatly increased due to the increased purity of the protein preparation loaded on the column.
  • the choice of capture column may also further improve the yield and effectiveness of the process.
  • cation exchange chromatography a high fraction of the remaining host cell proteins will pass through the column in the initial flow through, thereby leaving a high binding capacity of the resin for the protein of interest.
  • the recombinant fusion protein is subjected to DPPI-I treatment and subsequently purified.
  • An aspect of the invention relates to a pre-sequence comprising a peptide MXB(ZB)n, wherein B is a basic amino acid individually selected from Arg and Lys,
  • X is a dipeptide, a small amino acid or absent
  • Z is any amino acid
  • n is at least 2.
  • the use of at least three basic amino acid residues in the pre-sequence allows high expression while securing an increase of the pi of the protein which may help to solubilise the protein at low pH and at the same enables the protein to bind to a cation exchange resin.
  • the total length of the pre-sequence is at most 20 amino acid residues.
  • n ranges from 2 to at most 5.
  • the pre-sequences of present invention offer a method to elevate the pi of the recombinant fusion protein enabling pH precipitation of host cell proteins without significant loss of the recombinant protein.
  • An aspect of the invention relates to a recombinant fusion protein comprising a pre- sequence as defined herein fused to a protein.
  • An aspect relates to a DNA vector comprising DNA sequences as described herein.
  • the pre-sequences of the invention are useful in recombinant protein production as the pre-sequences may be removed to obtain a protein without an N-terminal methionine.
  • An aspect of the invention relates to a method for producing a protein with or without an N- terminal methionine comprising the steps of:
  • the pre-sequences of the invention add further advantages to the process of producing a protein of interest as the pre-sequences increase the overall isoelectric point of the recombinant fusion protein, which in turn allows 1 ) acidic pH precipitation of host cell protein from E. coli without significant loss of the protein of interest and secondly 2) purification hereof using cation exchange
  • the present invention relates to methods for improving expression and purification of recombinant proteins.
  • Recombinant expression of proteins allows production of various proteins in different hosts suitable for different proteins including heterologous expressions of for example human proteins in E. coli, yeast or mammalian cells as host cells.
  • the protein of interest may be produced by means of recombinant nucleic acid techniques.
  • a cloned nucleic acid sequence is modified to encode the desired protein. This modified sequence is then inserted into an expression vector, which is in turn transformed or transfected into host cells.
  • the DNA sequence encoding the protein is usually inserted into 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.
  • Standard methods involve cloning of the protein and subsequence expression in a suitable host.
  • a cloned wild-type nucleic acid sequence is obtained and if desired modified to include one or more mutation to encode a specific amino acid variant.
  • This sequence is then inserted into an expression vector, which is in turn transformed or transfected into a selected host cell.
  • the nucleic acid sequence or fragment encoding the protein may suitably be of genomic, cDNA or synthetic origin. Amino acid sequence alterations are accomplished by modification of the coding region by well-known techniques, such as by using quick change site-directed mutagenesis kit's.
  • 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 protein of interest followed by a terminator.
  • a secretory signal sequence To direct the protein into the secretory pathway of the host cells, a secretory signal sequence
  • signal peptide (also known as signal peptide) is useful and may be applied as an N-terminal extension of the protein.
  • a DNA sequence encoding the signal peptide is joined to the 5' end of the DNA sequence encoding the protein in the correct reading frame.
  • the signal peptide may be a peptide normally associated with the protein or may be from a gene encoding another secreted protein.
  • DNA sequence encoding the presequence of the invention is joined to the 5'end of the DNA sequence encoding the protein in the correct reading frame, whereby a single stretch of DNA encoding the recombinant fusion protein is obtained.
  • a pre-sequence or peptide tag may be used for improving the expression process or purification of a protein.
  • the DNA sequence coding for the protein is linked in-frame with codons encoding a pre-sequence which is cleaved of during the further processing.
  • the pre-sequence is thus linked to a heterogeneous protein and the resulting protein sequence is thus not found in nature.
  • the tag may additionally serve as a purification tag in the earlier processing of the protein.
  • a cleavable N- terminal pre-sequence may be used. This system can be used to provide native N-terminals of proteins that are post-translationally processed in vivo. It is none the less clear to the skilled person that cleavable pre-sequences can also be used for expressing variants of proteins. As the initial methionine is part of the pre-sequence, the protein produced after cleavage need not include a N- terminal methionine and basically any N-terminal amino acid residue can be positioned downstream of the pre-sequence.
  • the pre-sequence of the invention comprises at least 3 basic residues, such as His, Arg or Lys.
  • the pre- sequence comprises three or four Lys residues.
  • the pre-sequence comprise three Lys residues.
  • two basic residues are not sufficient to provide good solubility while too many basic residues may compromise the activity of the peptidase and thereby the removal of the pre-sequence in case this is desired. This is demonstrated in Example 4, where 6 or 7 Lys residues results in incomplete release of hGH by DPPI-I.
  • the length of the pre-sequence should be minimized not to influence productivity of the expression process.
  • the pre-sequence consists of at most 20 amino acid residues, such as at most 15, at most 10 at most 8 amino acid residues.
  • the pre-sequence according to the invention comprise at least 5 amino acid residues, such as at least 6 AA, such as at least 7 AA or such as at least 8 AA.
  • the pre-sequence consists of 3-20 amino acid residues, such as 5- 15 amino acid residues, such as 5-14 amino acid residues, such as 6-13 amino acid residues, such as 6-12 amino acid residues, such as 6-10 or such as 6-8 amino acid residues.
  • a small amino acid may be included after the initial Met residue.
  • the pre- sequence of the invention preferably comprises a small amino acid, such as Ala, Gly or Ser in the second position.
  • a di-peptide or no amino acid may be used in the position after the initator Met.
  • the pre-sequence according to the invention has the structure of MXB(ZB)n, wherein B is a basic amino acid individually selected from Arg and Lys, X is a dipeptide, a small amino acid or absent, Z is any amino acid and n is at least 2.
  • X is a di peptide selected from, HQ, HT, QH and QT or HT and QT. In an alternative embodiment X is a small amino acid or absent. In an embodiment X is selected from Thr, Ala, Gly, Val and Ser or Thr, Ala, Gly and Ser or Ala, Gly and Ser.
  • Z is not Glu, Asp, Arg, Lys, Pro or Cys.
  • Zs are individually selected from the group of: Gin, His, Ala, Leu, Val, Gly, Phe, Met, Ser and Thr.
  • Z's are individually selected from Ala, Ser, Gly, Val and Thr.
  • Z is individually selected from Met, Ser and Thr.
  • Z is individually selected from Ser and Thr.
  • the peptide comprises MXB(Z-
  • the peptide comprises MXB(Z-
  • the presequence comprises or consists of the peptide MXB(ZB)n, wherein B is selected from Arg and Lys, X is a small amino acid or absent and Z is selected from Ser and Thr, wherein n is at least 2.
  • B is Lys.
  • the pre-sequence comprises or consists of the peptide MXB(ZB)n, wherein B is Lys, X is as defined above and Z is independently selected from S and T. In a further such embodiment X is either absent or a small amino acid as defined above.
  • the pre-sequence has the structure MXK(ZK)n, wherein X is S or A or G or absent, and n is at least 2, such as 2-6 or 2-5.
  • the pre-sequence comprises or consists of the peptide MSK(TK) n wherein n is at least 2. In one embodiment the pre-sequence comprises or consists of the peptide MSKTKTK.
  • the pre-sequence comprises or consists of the peptide of MK(TK) n wherein n is at least 2. In one embodiment the pre-sequence comprises or consists of the peptide MKTKTK.
  • fusion proteins refer to a polypeptide that comprises or consists of two individually defined amino acid sequences which are linked by ordinary peptide bonds as obtained by continuous translation of an mRNA transcript.
  • An aspect of the invention relates to a recombinant fusion protein comprising a pre- sequence as defined above. The presequence is fused to a protein of interest.
  • a recombinant fusion protein defines a fusion of two amino acid sequence (the presequence and a protein of interest) which are not fused in nature.
  • the expression of a recombinant fusion protein is preceded by the creation of a recombinant vector linking DNA sequences encoding the pre-sequence and the protein of interest.
  • a protein encoded by a naturally occurring organism with an N-terminal amino acid sequence equal to the pre-sequence as defined herein is not considered a recombinant protein.
  • the recombinant fusion protein may be the final product needed while in other situations it may be considered an intermediate in production of the protein of interest which is to be obtained after removal of the presequence.
  • the pre-sequence is fused to the N- terminal of a protein of interest as is described further below.
  • the presequence is removable by use of a protease or peptidase. Due to the basic nature of the pre-sequence of the invention, the overall isoelectric point (pi) of the recombinant fusion protein may change compared to the isoelectric point (pi) of the protein alone.
  • the pi of a protein or a recombinant fusion protein may be calculated based on the primary amino acid sequence or measured using isoelectric focusing (IEF) or by isoelectric capillary electrophoresis using such as an ICE280 (Convergent) described in example 8.
  • IEF isoelectric focusing
  • ICE280 Convergent
  • the pi of the recombinant fusion protein is increased compared to the pi of the protein. In one such embodiment the pi is increased at least 0.5 pH unit. In an embodiment, the pi increase helps solubilize the recombinant fusion protein at low pH, e.g. the recombinant fusion protein may be soluble under conditions where the protein (without the presequence) is not soluble.
  • the pi of the recombinant fusion protein is around 0.8 pH unit higher than the pi of the protein when either calculated using GPMAW or measured using an ICE280 instrument (see example 8).
  • the protein of the present invention may be any protein of interest.
  • a protein is according to the present invention usually a polypeptide of at least 20 amino acid residues, such as at least 50 amino acid residues or such as at least 150 amino acid residues.
  • the protein may be of any origin e.g. encoded and/or expressed by any species or alternatively a protein only obtained by recombinant processes where a specific DNA sequence is designed to encode the amino acid sequence of interest.
  • the pre-sequence of the invention has particular advantages when a protein devoid of an N-terminal methionine is desired.
  • This can be such as a native/mature protein where an N-terminal peptide including the start-methionine is to be removed or a truncated protein where it is unattractive to include an N-terminal methionine in the final protein product.
  • a protein comprising an N-terminal methionine is to be produced, this may obviously be obtained by including a codon encoding Met in the relevant position.
  • the recombinant fusion protein includes a pre-sequence fused to a growth hormone.
  • the protein may advantageously have a pi between 4 and 7, such as a pi between 5 and 6 as calculated or measured using appropriate technologies such as GPMAW software or an ICE280 instrument.
  • Growth hormone for the purpose of purification as described herein below the protein may advantageously have a pi between 4 and 7, such as a pi between 5 and 6 as calculated or measured
  • Growth hormone is a polypeptide hormone secreted by the anterior pituitary in mammals.
  • GH is a protein composed of approximately 190 amino acid residues corresponding to a molecular weight of approximately 22kDa.
  • Mature human growth hormone consist of 191 amino acid (hGH, SEQ ID NO: 1 ) which is formed after cleavage of a 217 amino acid precursor protein where a signal peptide of 26 N-terminal amino acid residues is removed.
  • the sequence of hGH is
  • the protein is a growth hormone polypeptide.
  • the protein is human growth hormone or a variant thereof.
  • Variants of hGH of particular interest may be found as described in WO2010084173 and WO201 1089250.
  • a growth hormone variant may differ from human growth hormone by addition and/or deletion and/or substitution of at least one amino acid residue compared to the naturally occurring human growth hormone sequence.
  • the term is used for a growth hormone protein wherein one or more amino acid residues of the growth hormone sequence has/have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the growth hormone and/or wherein one or more amino acid residues have been added and/or inserted to the growth hormone.
  • a growth hormone variant according to the invention comprises less than 8 modifications (substitutions, deletions and/or additions) relative to human growth hormone. In one embodiment a growth hormone variant comprises less than 7 modifications (substitutions, deletions and/or additions) relative to human growth hormone. In one embodiment a growth hormone variant comprises less than 6 modifications (substitutions, deletions and/or additions) relative to human growth hormone. In one embodiment a growth hormone variant comprises less than 5 modifications (substitutions, deletions and/or additions) relative to human growth hormone. In one embodiment a growth hormone variant comprises less than 4 modifications (substitutions, deletions and/or additions) relative to human growth hormone.
  • a growth hormone variant comprises less than 3 modifications (substitutions, deletions and/or additions) relative to human growth hormone. In one embodiment a growth hormone variant comprises less than 2 modifications (substitutions, deletions and/or additions) relative to human growth hormone.
  • the growth hormone variant is at least 90, 92, 94, 96 or 98 % identical to human growth hormone identified by SEQ ID NO: 1.
  • the growth hormone variant is at least 95, 96, 97, 98 or 99 % identical to human growth hormone identified by SEQ ID NO: 1 .
  • a growth hormone variant comprises exactly 7 amino acid modifications. In one embodiment a growth hormone variant comprises exactly 6 amino acid modifications. In one embodiment a growth hormone variant comprises exactly 5 amino acid modifications. In one embodiment a growth hormone variant comprises exactly 4 amino acid modifications. In one embodiment a growth hormone variant comprises exactly 3 amino acid modifications. In one embodiment a growth hormone variant comprises exactly 2 amino acid modifications. In one embodiment a growth hormone variant comprises exactly 1 amino acid modifications.
  • An aspect of the invention relates to a nucleotide/DNA sequence or segment encoding the presequence or the recombinant fusion protein described herein above.
  • the pre-sequence or the recombinant fusion protein is encoded by a DNA segment or DNA sequence which in further embodiments may be connected with promoter and termination regions.
  • a further aspect of the invention relates to a recombinant vector hosting a DNA sequence encoding the pre-sequence or the recombinant fusion protein according to the invention.
  • the DNA sequence is usually inserted into 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.
  • 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 extra-chromosomal 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 recombinant fusion protein 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 a recombinant fusion protein will comprise a promoter capable of initiating and directing the transcription of a cloned DNA sequence/segment.
  • 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 a recombinant fusion protein 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.
  • Expression of recombinant fusion protein can aim 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.
  • the recombinant vector comprise a DNA sequence encoding the presequence of the invention alone or in fusion with a protein.
  • the vector may further comprise one or more of a replicator unite, a selectable marker, a promoter, a terminator and restriction sites.
  • the host cell into which a recombinant vector encoding the recombinant fusion protein is introduced may be any cell that is capable of expressing the protein either intracellular or extracellular. If posttranslational modifications are needed, suitable host cells include yeast, fungi, insects and higher eukaryotic cells such as mammalian cells including HEK cells are consider useful hosts cells. For multiple purposes also prokaryotic host cells such as E. coli are considered appropriate.
  • Host cells carrying a vector according to the invention are further embodiments of the invention.
  • 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 ef 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, e.g. the synthetic promoter used to express hGH in Dalb0ge et al, 1987, Biotechnology 5, 161-164. Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook ef 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 STM, all from E. coli.
  • signal peptides can be created cfe 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 strong terminators for transcription are the aspartase aspA as in the Thiofusion Expression System, the T7 gene 10 terminator in the pET vectors and the terminators of the ribosomal RNA genes rrnA, rrnD.
  • 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 e.g. E. coli B BL21 DE3 derived strains such as a strain with deletion of the 2 D,L-alanine racemase genes Aalr, AdadX, and deletion of the Group II capsular gene cluster A(kpsM-kpsF), specific for E. coli B and often associated with pathogenic behaviour.
  • the deletion of the Group II gene cluster brings E.
  • the pre-sequence of the invention leads to a highly effective method for producing recombinant proteins with or without an N-terminal methionine.
  • the invention relates to a method for producing a protein comprising the steps of:
  • the method is for obtaining a protein without an N-terminal methionine.
  • the pre-sequence is cleavable allowing the presequence to be removed by enzymatic cleavage.
  • the pre-sequence serves a substrate of a proteinase or peptidase.
  • the pre-sequence provides a substrate of an aminopeptidase, such as a diaminopeptidase such as dipeptidyl aminopeptidase peptidase I.
  • the pre-sequence is removed using DPPI.
  • the protein is obtained by removing the pre-sequence using a EC 3.4.14.1 DPPI I, such as an DPPI from a vertebra species.
  • the method may include a step of removing host cell proteins.
  • the method may include a precipitation step.
  • the pi of the recombinant fusion protein is increased and a precipitation performed at a pH around one pH unit below the pi of the recombinant fusion protein is effective to precipitate host proteins while leaving the recombinant fusion protein soluble.
  • the method includes precipitation at a pH at least one pH unit below the pi of the recombinant fusion protein. In one embodiment the precipitation is performed at a pH around 1.5 pH units below the pi of the recombinant fusion protein. As mentioned previously the pi of the recombinant protein may be either calculated using appropriate software or measured experimentally.
  • the precipitation is performed at a pH in the range of 4-6, such as at a pH of approximately 5.
  • the precipitation step is performed prior to removal of the presequence.
  • the precipitate is preferably removed using centrifugation and/or filtration.
  • the method may include a step of purifying the recombinant fusion protein. Depending on the nature of the recombinant fusion protein various methods are available to the person skilled in the art.
  • the method may include a capture step, such as a chromatography capture step, such as cation exchange chromatography (CIEX).
  • a capture step such as a chromatography capture step, such as cation exchange chromatography (CIEX).
  • CIEX cation exchange chromatography
  • the recombinant fusion protein is expressed in E. coli.
  • initial/N-term Met (of the pre-sequence) is removed in vivo by endogenous enzymes of the host cell.
  • initial/N-term Met (of the pre-sequence) is removed during E. Coli expression. Following removal of the initial Met the remaining pre-sequence is removed by DPPI.
  • the cleavage of the presequence can be performed based on common general knowledge and optimized by the skilled person depending on specific sequence used and the protein of interest.
  • the recombinant fusion protein is treated with DPPI over night at room temperature using a protein:DPPI w/w ratio of 1000: 1 at pH 8.
  • the ratio of protein:DPPI is 2000:1 (w/w) and the reaction performed at pH 6-6.5 at room temperature overnight.
  • the recombinant fusion protein is treated with DPPI for one hour at room temperature overnight using a GH:DPPI w/w ratio of 1000: 1 at pH 6.
  • E. coli is the host.
  • the E. coli host is a K12 derived strain, BL21 (DE3) or a BL21 (DE3) derived strain.
  • Standard methods of expressing the recombinant fusion proteins may be employed.
  • the E. coli host is cultured at 37 °C before induction.
  • expression is induced at a temperature below 37 °C, such as below 32 °C, such as below 28 °C, such as around 25 °C or such as around 22 °C.
  • expression is induced using an IPTG concentration below 0.5 mM, such as 0.4 mM or 0.2 mM.
  • the induction period is from 12-24 hours, such as 12-18 hours, such as around 13-16 hours.
  • the expression is performed under high cell-density fermentation as fed- batch in fermentation tanks of 5-1000 L, such as 5 L, such as 20 L, such as 100 L or such as 1000 L.
  • the expression is performed under high cell-density fermentation as fed- batch in fermentation tanks of more than 1000 L, such as 2000 L, such as 5000 L, such as 10000 L.
  • the expression is performed in low cell-density fermentation using shake flasks. In one embodiment the fermentation is performed at a pH of 6-8, such as around 7.
  • the invention further relates to a method for preparing a pharmaceutical product.
  • the protein obtained by the method described herein above may accordingly be used in a pharmaceutical product.
  • the pharmaceutical product may be a formulation comprising the protein as well as one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and/ or a surfactant, as well as various combinations thereof.
  • the use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person.
  • the formulations may be prepared using standard procedures know in the art. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • An aspect of the invention relates to a method for preparing a pharmaceutical composition including a protein wherein the method comprises the steps of:
  • the presequence, the recombinant fusion protein and the protein may be as described herein above, such that the presequence is directly fused to a therapeutic protein as in the example of human growth hormone as described here in below.
  • the method may include further steps, including one or more of the steps described herein above. Also subsequent steps relating to modification of the protein, such as chemical modification of the protein prior to preparation of the pharmaceutical composition are contemplated.
  • a pre-sequence comprising a peptide MXB(ZB)n, wherein
  • B is a basic amino acid individually selected from Arg and Lys,
  • X is a dipeptide, a small amino acid or absent
  • Z is any amino acid
  • n is at least 2.
  • pre-sequence according to embodiment 1 wherein the pre-sequence consists of at most 20 amino acid residues, such as at most 15, at most 10 at most 8 amino acid residues.
  • the pre-sequence according to any of the previous embodiments wherein the pre-sequence comprises at least 5 amino acid residues, such as at least 6 AA, such as at least 7 AA or such as at least 8 AA.
  • the pre-sequence according to any of the previous embodiments wherein the peptide comprises MXB(ZB-
  • Zs are not Glu, Asp, Arg, Lys, Pro or Cys.
  • to n B)n and Z-i t0 n are individually selected from S and T.
  • n 2-6,2-5, 2-4 or 2-3.
  • X is a small amino acid selected from the group of: Ala, Gly, Ser, Val and Thr.
  • pre-sequence according to any of the previous, wherein the pre-sequence is selected from the group consisting of: MSK(ZK)n, MAK(ZK)n and MGK(ZK)n, wherein n is at least 2 and Z is selected from the group of Ser and Thr. 18.
  • pre-sequence according to embodiment 21 wherein the pre-sequence consists of the peptide MKSKTKSKTKSK, MKSKTKSKTKSKTK, MKSKSK or MKTKTK.
  • a recombinant fusion protein comprising a pre-sequence as defined in any of embodiments 1-23 and a protein.
  • a recombinant vector comprising the DNA sequence of any of the embodiments 36-38. 40.
  • a method for producing a protein comprising the steps of:
  • the peptidase is an aminopeptidase.
  • the peptidase is a diaminopeptidase.
  • the peptidase is dipeptidyl aminopeptidase I (DPPI).
  • DPPI dipeptidyl aminopeptidase I
  • the dipeptidyl aminopeptidase I (DPPI) is an EC 3.4.14.1 dipeptidyl-peptidase I.
  • the dipeptidyl aminopeptidase I (DPPI) is from a vertebra species.
  • the method according to any of the embodiments 48-51 wherein the precipitate is removed by centrifugation and/or filtration.
  • a method for preparing a pharmaceutical composition including a protein wherein the method comprises the steps of:
  • pET1 1d-MEAE-hGH was constructioned by insertion of MEAE-hGH into the Nco I and BamH I sites of pET1 1d (Novagen).
  • Site directed mutagenesis was used to create different presequences based on pET1 1d-MEAE-hGH.
  • the primers used are identified in table 1. The reactions were performed according to manufactures instructions (QuickChange Lightning Site-Directed Mutagenesis, Stratagene). Pre-sequence DNA of Primer sequence (5') Primer sequence (3') presequence
  • MKTKTK atgaaaaccaaaacc CCATGAAAACCAAAAcCAA GTTGGGAATTTGCTTTTGC (SEQ ID NO 5) aaa ATTCCCAACCATTCCCTTA I I I CATGGTATATCTCCT
  • MKSKTKSKTKS atgaaaagcaaacc CCATGAAAAGCAAAACCA GCTTTTGGTTTTGCTTTTG KTK (SEQ ID NO aaaagcaaaaccaa AAAGCAAAACCAAAAGCA GTTTTGCTTTTCATGGTAT 7) aagcaaaaccaaa AAACCAAATTCCCAACCAT ATCTCCTTCTTAAAG (SEQ ID NO aaaagcaaaccaa AAACCAAATTCCCAACCAT ATCTCCTTCTTAAAG (SEQ ID NO aaagcaaaccaa AAACCAAATTCCCAACCAT ATCTCCTTCTTAAAG (SEQ ID NO aaagcaaaccaa AAACCAAATTCCCAACCAT ATCTCCTTCTTAAAG (SEQ ID NO aaagcaaaccaa AAACCAAATTCCCAACCAT ATCTCCTTCTTAAAG (SEQ ID NO aaagcaaaccaa AAACCAAATTCCCAACCAT ATCTCCT
  • Each of the constructs were transformed into BL21 (DE3) to evaluate expression levels and solubility.
  • Colonies were used to inoculate 500 mL LB medium for expression. Cultures were incubated at 37°C with shaking at 220 rpm until OD 60 o 0.6 was reached. Induction was performed using 0.1 mM IPTG at 22°C or 25°C overnight. Cells were harvested by centrifugation at 6000 rpm for 20 min and suspended in cell lysate buffer consisting of 50 mM Na-phosphate, 0.1 % Tween-20, 5 mM EDTA pH 8.5. Cells were homogenised by sonication and urea and cystamine added to final concentrations of 2 M and 2 mM, respectively. The cell lysates were incubated overnight at 4 °C. To compare expression levels of the different constructs, total cell lysate, supernatant and pellet after lysis in cell lysate buffer was analysed by SDS page (data not shown)
  • Fermentation was carried out in 1 L Q plus fermenter containing 0.6L fermentation medium. The medium was inoculated to OD 0.1 with fresh E. coli culture at OD 3-6 (in mid log phase) in EC1 medium. Batch culture was carried out at T 37°C and pH 7.0. Fed-batch was initiated after glucose was consumed which was indicated by a sharp p0 2 increase. After 10h fed batch phase, temperature was reduced to 25°C and induction was started by adding 0.2 mM IPTG. During the whole fermentation, pH was controlled at 7.0 by adding 8% NH 3 H 2 0 and p0 2 was kept above 20%. The feeding for fed-batch phase varied between 4.50 and 20.08 g Glucose/L/h. During induction, feeding was kept constant at 8.5 g Glucose/L/h.
  • Example 4 Evaluation of basic pre-sequences (solubility, purification and digestion of pre-sequence) Each construct was expressed in 1 liter of fermentation media (as described in Example 3).
  • the constructs with two Lys (K) residues provided good expression but approximately 1/3 of the expressed protein was insoluble while all of MKTKTK-, MKSKTK-, MKSKTKSKTKSKTK and MKSKTKSKTKSK gave rise to a high level soluble protein as evaluated by SDS PAGE of total lysate and supernatant and pellet after centrifugation of lysed samples.
  • Cells were cultured and expression induced as described above (Example 3). The harvested cells were suspended in 50 mM Na-phosphate, 0.1 % Tween-20, 5 mM EDTA pH 8.5 for homogenisation. Homogenised cells were incubated overnight after addition of 2 M urea, 2 mM cystamine (final concentrations). Host cell proteins were precipitated by pH adjustment to 5.0 using 2 M citric acid. Before capture, clarification was performed by centrifugation followed by filtration.
  • Cation exchange capture was run on POROS 50 HS at pH 5.0 using linear salt gradient elution. Eluting protein was detected by absorbance at 254 and 280 nm. Fractions were collected throughout the whole elution gradient and fractions were subsequently pooled to separately collect the different peaks observed in the chromatogram. The purity prior to and after cation exchange capture was analysed by RP-HPLC and was found to increase from 35% to 85%.
  • Example 6 Further optimisation of pre-sequence A minor degree of methylation (around 10 %) was observed on the MKTKTK presequence, which has the disadvantage that protein with methylation of the N-terminal methionine cannot be efficiently digested by the DPPI enzyme.
  • a further series of constructs introducing one or two amino acid residues between M and K was prepared using site directed mutagenesis as described above.
  • Primer sequence 5'
  • Primer sequence 3'
  • sequence g MHTKTKTK atgcatactaaaaccaaac CTTTAAG AAG GAG ATA GTTGGGAATTTGGTTTT (SEQ ID NO caaa (SEQ ID NO 19) TACCATGCATACTAAA GG I I I AGTATGCATG 8) ACCAAAACCAAATTCC GTATATCTCCTTCTTAA
  • CAAC (SEQ ID NO 30) AG (SEQ ID NO 41 ) h) MQTKTKTK( atgcagactaaaaccaaaa CTTTAAG AAG GAG ATA GTTGGGAATTTGGTTTT SEQ ID NO ccaaa (SEQ ID NO 20) TACCATGCAGACTAA GG I I I I AGTCTGCATG
  • MAKTKTK atggctaaaaccaaacca CTTTAAG AAG GAG ATA GTTGGGAATTTGGTTT SEQ ID NO aa (SEQ ID NO 21 ) TACCATGGCTAAAAC TGG I I I I AGCCATGGT 10) CAAAACCAAATTCCCA ATATCTCCTTCTTAAAG
  • the expressed proteins were purified as described in example 5 followed by mass spectrometry LC/MS analysis of the intact mass to evaluate level of methylation and removal of the N- terminal Methionine by endogenous E. coli enzyme methionine amino peptidase. Protein samples were subjected to digestion by DPPI followed by LC/MS analysis to evaluate the amount as area under the curve of the LC chromatogram of digested versus undigested protein.
  • Results are summarised in table 4 below. All construct expressed intermediate to high level of protein. Methylation was decreased for all constructs compared to MKTKTK except for M-AKTKTK. The three constructs including a small amino acid residue (A, G or S) were all shown to be substrates of in vivo removal of the initial methionine, whereas DPPI digestion of M-GKTKTK was inferior to digestion of MKTKTK.
  • MKTKTK-hGH (Q84C, Y143Q, L101 C) in example 5 above was tested for the constructs MKTKTK- and MSKTKTK-hGH and no significant differences were observed. Recovery and capture ran as expected and with similar yields and purities.
  • M-SKTKTK-hGH variant no methionine-containing protein could be detected by LC-MS analysis and no methylation of the N-terminal Ser residue was observed.
  • Example 8 Determination of isoelectric point
  • the theoretical isoelectric point of GH and a variant including the MSKTKTK pre-sequence were calculated using GPMAW software version 9.51 from Lighthouse Software.
  • the isoelectric point of GH with and without the MSKTKTK presequence was determined experimentally using an ICE280 Instrument from Convergent coupled to a PrinCe microinjector.
  • the experimental result confirms the increase in pi of approximately one unit when comparing hGH to MSKTKTK-hGH.

Abstract

L'invention concerne de nouvelles pré-séquences qui facilitent l'expression et la purification efficaces de protéines recombinantes. En particulier, les pré-séquences et les procédés associés permettent la purification d'une protéine dépourvue de méthionine N-terminale.
PCT/EP2015/069846 2014-09-01 2015-08-31 Pré-séquences basiques riches en lysine WO2016034534A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1364900A (zh) * 2001-01-10 2002-08-21 上海博德基因开发有限公司 一种新的多肽——人 atp依赖的丝氨酸蛋白水解酶47.19和编码这种多肽的多核苷酸
WO2008003750A2 (fr) * 2006-07-07 2008-01-10 Novo Nordisk Health Care Ag Nouveaux conjugués de protéines et leurs procédés de préparation
WO2008104513A1 (fr) * 2007-03-01 2008-09-04 Novo Nordisk A/S Expression de protéines dans e.coli

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Publication number Priority date Publication date Assignee Title
CN1364900A (zh) * 2001-01-10 2002-08-21 上海博德基因开发有限公司 一种新的多肽——人 atp依赖的丝氨酸蛋白水解酶47.19和编码这种多肽的多核苷酸
WO2008003750A2 (fr) * 2006-07-07 2008-01-10 Novo Nordisk Health Care Ag Nouveaux conjugués de protéines et leurs procédés de préparation
WO2008104513A1 (fr) * 2007-03-01 2008-09-04 Novo Nordisk A/S Expression de protéines dans e.coli

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DATABASE Geneseq [online] 24 March 2003 (2003-03-24), "ATP dependent serine protein hydrolase 47.19 N-terminal peptide.", XP002738570, retrieved from EBI accession no. GSP:ABP58709 Database accession no. ABP58709 *
DATABASE UniProt [online] 24 July 2013 (2013-07-24), "SubName: Full=Uncharacterized protein {ECO:0000313|EMBL:EOQ11192.1};", XP002738571, retrieved from EBI accession no. UNIPROT:R8TWX8 Database accession no. R8TWX8 *
DATABASE UniProt [online] 25 January 2012 (2012-01-25), "SubName: Full=Uncharacterized protein {ECO:0000313|EMBL:CCE65183.1};", XP002738572, retrieved from EBI accession no. UNIPROT:G8BZ55 Database accession no. G8BZ55 *
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