[go: up one dir, main page]

WO2007068053A1 - Production de protéines par voie recombinante - Google Patents

Production de protéines par voie recombinante Download PDF

Info

Publication number
WO2007068053A1
WO2007068053A1 PCT/AU2006/001898 AU2006001898W WO2007068053A1 WO 2007068053 A1 WO2007068053 A1 WO 2007068053A1 AU 2006001898 W AU2006001898 W AU 2006001898W WO 2007068053 A1 WO2007068053 A1 WO 2007068053A1
Authority
WO
WIPO (PCT)
Prior art keywords
igf
construct
seq
growth factor
sequence
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/AU2006/001898
Other languages
English (en)
Inventor
Geoffrey Leonard Francis
Graham Dean Hobba
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes Biopharma AU Ltd
Original Assignee
Gropep Ltd
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 Gropep Ltd filed Critical Gropep Ltd
Publication of WO2007068053A1 publication Critical patent/WO2007068053A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to methods of recombinant production of proteins, such as growth factors.
  • the invention relates to improvements in methods for the production of growth factor proteins as fusion proteins .
  • the protein is a growth factor such as IGF or EGF.
  • IGFs Insulin-like growth factors
  • IGF-I is a small protein which has been shown to stimulate the growth of a wide range of cells in culture.
  • IGF-Is The sequences of some mammalian IGF-Is and IGF-IIs are as follows:
  • Cow AYRPSETLCGGELVDTLQFVCGDRGFYFSRPSSRINRRSRGIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 9)
  • IGF-I has a wide variety of potential applications, including its use to:
  • IGF-I has also been widely used as a supplement in serum-free cell culture media. Analogues of IGF-I and IGF-II are described in US Patent No. 5,330,971, the entire disclosure of which is incorporated herein by this cross-reference.
  • LONG 18 R 3 IGF-I (MetpGH (1-11) VNR 3 IGF-I)
  • LONG 8 R 3 IGF-I is significantly more potent than human IGF-I in vitro.
  • the enhanced potency is due to decreased binding of LONG 8 R 3 IGF- I to IGF binding proteins, which normally inhibit the biological actions of IGFs.
  • LONG 0 R 3 IGF-I is currently used in the commercial manufacture of 7 therapeutic agents approved by the US Food and Drug administration.
  • IGF-I human des (1-3) IGF-I (SEQ ID NO: 12) . This is a 67 amino acid analogue of IGF-I which lacks the N-terminal tripeptide Gly-Pro-Glu. Des(l- 3) IGF-I is more potent than IGF-I in vitro and in vivo.
  • porcine growth hormone-derived fusion partner has been shown to promote the correct folding of the fusion protein into its preferred biologically active form (Milner et al . , (1995), Biochemical Journal 308:865-871).
  • Methods for the recombinant production of IGF-I (SEQ ID NO: 13), IGF-II (SEQ ID NO: 12) and analogues thereof, such as LONG 0 R 3 IGF-I and des (1-3) IGF-I include those methods described in Francis et al . , Milner et al . and US Patent No. 5,330,971.
  • leader sequence is a polypeptide having growth hormone activity or a fragment thereof.
  • the polypeptide comprising the leader sequence is referred to as the fusion partner, and the IGF is referred to as the fusion protein.
  • the leader sequence is homologous to the first 1-191 N-terminal amino acids of methionine porcine growth hormone (metpGH or MpGH) . This leader sequence is set out in SEQ ID NO: 14.
  • the fusion protein may optionally further comprise a chemical or enzymatic cleavage site between the leader sequence and the IGF so as to allow the release of mature IGF or analogue following cleavage of the fusion protein. Without this leader sequence, yields of the IGF following cell culture are very low, and inadequate for manufacture of
  • US Patent No. 5,330,971 also discloses additional leader sequences, including: MFPAMPLSSLF- (SEQ ID NO: 15); MFPAMPLSSLFVN- (SEQ ID NO:16);
  • leader sequences contain an initiating methionine at their N-terminal end, followed by a sequence homologous to the first 10 amino acids of pGH.
  • SEQ ID NOs 16, 17, and 18 also contain other amino acids, as indicated above. These are encoded by the Hpal restriction site.
  • the inventors have recognized a need in the art for improved methods for the commercial-scale production of proteins, such as IGFs and other growth factors, which provide protein yields which are higher than those which can be achieved with presently available techniques.
  • IGF or epidermal growth factor (EGF) is expressed so that , it is linked to a leader sequence which comprises the amino acid sequence LSTQ (SEQ ID NO: 19), yields of recombinant protein are increased compared to yields obtained using prior art methods.
  • LSTQ acts as a cleavage site for ⁇ -lytic proteases, which are described in Haggett et al (1994), Arch. Biochem. Phys . 314 (1) 132-141.
  • the invention provides: 1. A peptide of amino acid sequence LSTQ (SEQ ID NO: 19) .
  • a peptide of the amino acid LSTQ (SEQ ID NO: 19) in a leader sequence to increase expression of a recombinant growth factor.
  • a fusion protein comprising: a) a peptide leader sequence of up to 200 amino acids comprising LSTQ (SEQ ID NO: 19); and b) a polypeptide growth factor.
  • a method of increasing the yield of a recombinant protein comprising the steps of:
  • a fusion protein comprising a peptide leader sequence of up to 200 amino acids containing LSTQ (SEQ ID NO: 19) and an amino acid sequence encoding a growth factor.
  • a construct comprising a first nucleic acid encoding a peptide leader sequence comprising the amino acid sequence LSTQ (SEQ ID N0:19), a second nucleic acid encoding a cleavage site and a third nucleic acid encoding a growth factor, in which said first, second and third nucleic acids are consecutive .
  • the invention provides a polypeptide fusion protein or construct (hereafter referred to as "the construct") comprising: a) a peptide or polypeptide leader sequence comprising the amino acid sequence LSTQ (SEQ ID NO: 19); and b) a polypeptide growth factor.
  • the growth factor is selected from the group consisting of insulin-like growth factor, epidermal growth factor (EGF) , transforming growth factor (TGF ⁇ ) , including TGF ⁇ l, TGF ⁇ 2, and TGF ⁇ 3 , fibroblast growth factor (FGF) , platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) .
  • EGF epidermal growth factor
  • TGF ⁇ transforming growth factor
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • the amino acid sequence for the growth factor is homologous to that of the human growth factor or a biologically active fragment thereof.
  • the construct of the invention exhibits growth factor activity.
  • the polypeptide growth factor is an IGF which stimulates protein synthesis in rat L6 myoblasts . Methods for assaying this activity are widely known in the art, and are described in G. L. Francis et al . Biochem. J. 233, 207-213, (1986), the entire disclosure of which is incorporated herein by this cross-reference.
  • the IGF is a biologically active fragment, functional analogue or derivative of IGF- I or IGF-II.
  • the IGF is selected from the group consisting of: [Arg] 3 IGF-I:
  • IGF-II AYRPSRTLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLET YCATPAKSE (SEQ ID NO: 21); des (1-3) IGF-I: TLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYC APLKPAKSA (SEQ ID NO: 12); and des (1-6) IGF-II:
  • the IGF is des (1-3) IGF-I (SEQ ID NO: 12) .
  • the leader sequence consists essentially of 4 to approximately 200 amino acids.
  • the leader sequence comprises a polypeptide, or a fragment thereof, which has growth hormone activity.
  • the leader sequence comprises a peptide sequence which is homologous to the first n to 191 N-terminal amino acids of methionine porcine growth hormone (metpGH or MpGH) , or an N-terminal fragment thereof, in which n is an integer between 1 and 190. In some embodiments n is i, 2, 3, 4, 5, 6, 7, 8, 9,
  • the leader sequence comprises a polypeptide which is useful in the expression of one or more other growth factors.
  • examples of other fusion partners used in the expression of growth factors include (a) Uhlen et al . (1992) Curr. Opin.
  • the peptide leader sequence in (a) is an amino acid sequence selected from the group consisting of: LSTQ (SEQ ID NO: 19) ; VNLSTQ (SEQ ID NO:23);
  • MFPAMPLSSLFLSTQ SEQ ID NO: 24
  • MFPAMPLSSLFVNLSTQ SEQ ID NO: 25
  • MFPAMPLSSLFVNGLSTQ SEQ ID NO: 26
  • MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNGLSTQ (SEQ ID NO: 29) .
  • LSTQ portion of the peptide leader sequence is linked consecutively to the growth factor.
  • the construct may optionally further comprise a chemical or enzymatic cleavage site between the leader sequence and the growth factor so as to allow the release of mature growth factor following cleavage of the construct, and /or one or more amino acids encoded by a restriction enzyme recognition site.
  • the construct comprises the amino acid sequence VN, which is encoded by the Hpal restriction enzyme recognition site.
  • the construct is selected from the group consisting of: MFPAMPLSSLFLSTQ-des (1-3) IGF-I (SEQ ID NO:30);
  • MFPAMPLSSLFVNLSTQ-deS (1-3) IGF-I (SEQ ID N0:31); MFPAMPLSSLFVNGLSTQ-des (1-3) IGF-I (SEQ ID NO:32); MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQLSTQ ⁇ des(l- 3) IGF-I (SEQ ID NO: 33); MFPAMPLSSLFANAVLRAQHLHQLAADTYYKEFERAYIPEGQRYSIQVNLSTQ- des (1-3) IGF-I (SEQ ID NO: 34); and
  • the construct is: MFPAMPLSSLFVNLSTQTLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIV DECCFRSCDLRRLEMYCAPLKPAKSA (SEQ ID NO: 36) .
  • the construct is metpGH(l- 11)VNLSTQdes (1-3) IGF-I (SEQ ID NO:37).
  • the polypeptide is EGF or an analogue or derivative thereof which retains at least one biological activity of EGF (for the amino acid sequence of EGF, see Bell et al . ; Nucleic Acids Res. 14 (21), 8427- ⁇ 8446 (1986) ; for a review of EGF activities and methods for their assay, see Jost et al . Eur J Dermatol. 2000 10 (7) :505-10) .
  • the peptide leader sequence in (a) does not comprise either of the amino acid sequences FAHY (SEQ ID NO:38) or GFAHY (SEQ ID NO:39).
  • the construct is substantially isolated.
  • it may be substantially isolated from a cell culture medium following recombinant production of the construct .
  • the invention provides an antibody against a construct according to the invention. It will be clearly understood that specific polyclonal or monoclonal antibodies against the constructs of the invention may readily be raised using methods routine in the art. These antibodies may be directed to an epitope comprising the tetrapeptide LSTQ (SEQ ID NO: 19), and may be used in assays such as immunoassays for the construct of the invention.
  • the invention provides an isolated nucleic acid molecule which encodes a construct according to the invention.
  • the nucleic acid may comprise nucleotide sequences of human origin or synthetic origin, and may be single-stranded or double-stranded DNA.
  • the nucleic acid may have codons for optimized expression in a desired host organism, such as E. coli.
  • the nucleic acid molecule is cDNA or RNA.
  • the invention provides a vector comprising a nucleic acid molecule according to the invention.
  • the vector may comprise a first nucleic acid molecule encoding the construct and a second nucleic acid molecule encoding a desired polypeptide.
  • the desired polypeptide may be a therapeutically useful protein.
  • the vector may be of any suitable type, and may be an expression vector or a plasmid.
  • the plasmid pGHXSC.4 may be used; this plasmid comprises the pGH coding region.
  • the invention provides a host cell transformed by a vector according to the invention.
  • the host cell is a prokaryotic cell.
  • One useful host cell is E. coli, for example JE?. coli strain JMlOl.
  • the host cell may be a eukaryotic cell, such as a mammalian cell.
  • the mammalian cell is a CHO cell, Per.C ⁇ TM cell, HEK293 cell, Vero cell, or MDCK cell, or any fibroblast cell line.
  • the host cell may be a yeast, such as Saccharomyces cereviseae or Pichia pastoris.
  • the invention provides a process for the production of a construct according to the invention, which process comprises:
  • the invention provides a process for the production of a construct according to the invention, comprising the steps of:
  • the unicellular organism is a prokaryotic organism.
  • the prokaryotic organism may, for example, be a bacterial strain, such as a strain of E. coli.
  • the E. coli strain JMlOl is one suitable unicellular organism.
  • the process may further include the step of cleaving the construct at a cleavage site between the leader sequence and the growth factor sequence to release the growth factor polypeptide, which may then be isolated. It is known from US Patent No. 5,330,971 that cleavage to remove the growth hormone-derived leader sequence may not be required to achieve adequate biological activity if this leader sequence is small. For example, where the leader sequence is approximately 10 amino acids, then cleavage of the leader sequence may not be required. The person skilled in the art will readily be able to determine whether cleavage is required in any specific leader sequence-growth factor combination, using routine methods .
  • the invention provides a method for culturing mammalian cells, comprising culturing the cells under cell growing conditions in a cell culture medium comprising a construct according to the invention in which the growth factor is IGF.
  • the medium suitably also comprises
  • the polypeptide is at a concentration of between 10-100 ng/ml .
  • the polypeptide may be at a concentration of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng/ml.
  • the medium does not contain serum.
  • each component of the medium is obtained from a source other than directly from an animal source.
  • the medium may further comprise one or more of non-ferrous metals, vitamins, transition metals, carrier proteins such as albumin or cofactors.
  • the cells may comprise an IGF receptor.
  • the cells may comprise an insulin receptor.
  • the mammalian cell is a CHO cell, Per.C6TM cell, HEK293 cell, Vero cell, MDCK cell, or fibroblast cell line.
  • the invention provides a method for culturing mammalian cells, the method comprising culturing and growing the cells in the absence of serum, in a medium comprising, per litre of medium:
  • amino acid selected from the group consisting of L-alanine, L- arginine, L-asparagine, L-aspartic acid, L-cystine, L- glutamic acid, glycine, L-histidine, L-isoleucine, L- leucine, L-lysine,
  • the invention provides a composition comprising a construct according to the invention, together with a pharmaceutically, veterinarily or cell culture acceptable carrier.
  • the composition is in dry powder form, and may further comprise a bulking agent.
  • the composition comprises a polypeptide according to the invention formulated in acid.
  • the acid is acetic acid or hydrochloric acid.
  • the polypeptide is formulated in 100 mM acetic acid or 10 mM hydrochloric acid.
  • the invention provides a cell culture system, comprising (a) a fermenter adapted for mammalian cell culture;
  • a mammalian cell culture medium comprising a construct according to the invention.
  • the mammalian host cell may be engineered to express both a desired protein such as a protein therapeutic agent and a construct according to the invention, which stimulates the proliferation of the mammalian host cell and increases yields of the desired protein.
  • the invention provides a cell culture system comprising: (a) a fermenter adapted for mammalian cell culture;
  • the construct comprises an IGF sequence and exhibits growth factor activity, which stimulates the proliferation of the mammalian host cell, increases cell survival, prolongs cell viability, and/or increases yield of the protein.
  • the cell culture medium comprises water, an osmolality regulator, a buffer, an energy source, at least one additional amino acid and an inorganic, organic or recombinant iron source.
  • the host cell is a CHO cell
  • the desired polypeptide is an anti-inflammatory protein
  • the construct is:
  • the invention provides a kit comprising a liquid composition or dry powder composition comprising a construct according to the invention sealed in a vial, cartridge or container.
  • the kit may be labelled by, or accompanied with, instructions for use in mammalian cell culture.
  • the instructions specify that the contents are not suitable for human therapeutic use .
  • the invention provides a composition for the treatment of a protein accumulation deficiency condition or protein loss in a mammal, comprising an effective amount of a construct according to the invention, together with a pharmaceutically acceptable diluent, carrier or excipient, in which the construct exhibits insulin-like growth factor activity.
  • the composition comprises the construct in an amount sufficient to provide a dose of approximately 0.01 to 10, preferably 0.1 to 1 mg/kg body weight/day.
  • the construct may be present in a unit dosage form in amounts from approximately 0.02 to 2000 mg.
  • Slow- release pellet implants as provided in conventional practice, are the preferred method of administration to animals .
  • the invention provides a method of treatment of a condition selected from the group ⁇ consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer, cystic fibrosis, Duchenne muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, polymyositis and other myopathies; acute conditions associated with poor nitrogen status including, but not limited to, burns, skeletal trauma and infection; or to promote growth, improve nitrogen status and/or to treat catabolic disorders in infants or premature babies, in a subject in need of such treatment, comprising the step of administering an effective amount of a construct according to the invention which exhibits insulin-like growth factor activity to a mammal in need of such treatment .
  • the invention provides the use of a construct according to the invention which exhibits insulin-like growth factor activity in the manufacture of a medicament for the treatment of a condition selected from the group consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer, cystic fibrosis, Duchenne muscular dystrophy, Becker dystrophy, autosomal recessive dystrophy, polymyositis and other myopathies; acute conditions associated with poor nitrogen status including, but not limited to, burns, skeletal trauma and infection; or for promoting growth, improving nitrogen status and/or treatment of catabolic disorders in infants or premature babies .
  • a condition selected from the group consisting of protein accumulation deficiency condition or protein loss; chronic growth disorders, including growth hormone deficiency and somatomedin deficiency; disorders associated with insufficient growth or tissue wasting, including, but not limited to, cancer,
  • the mammal may be a human, or may be a domestic, companion or zoo animal. While it is particularly contemplated that the constructs of the invention and growth factors expressed using the leader sequences of the invention are suitable for use in medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates. It will be appreciated that there is a very high degree of sequence conservation in growth factor sequences between mammalian species. While the invention is described in detail with reference to IGF and EGF, it is to be clearly understood that it is contemplated that the invention described herein is also applicable to other growth factors.
  • Figure 1 shows dose response curves illustrating the ability of (A) LONG 0 R3IGF-I (GroPep Ltd) and (B) metpGH(l- 11) WLSTQdes(1-3) IGF-I to stimulate the growth of L6 myoblasts in culture.
  • a polypeptide includes a plurality of such polypeptides
  • a reference to “an amino acid” is a reference to one or more amino acids.
  • IGF-I IGF-I, IGF- II and biologically active fragments, functional analogues or derivatives thereof are considered to be equivalent, and are referred to collectively as "IGF” or "IGFs” .
  • ⁇ methionine porcine growth hormone (metpGH) ' means porcine growth hormone, in which methionine has been substituted for the amino acid normally at the N-terminus.
  • fragment means a molecule derived from a growth factor, in which the molecule retains at least one biological function or activity as that of the original growth factor.
  • consisting essentially of means that other amino acids can be added to the amino or carboxyl terminal of the peptide LSTG without affecting the ability of LSTG to increase protein expression.
  • amino acid or "amino acid residue”, as used herein, refers to naturally occurring L amino acids.
  • amino acids The commonly used one-and three-letter abbreviations for amino acids are used herein (Bruce Alberts et al . , Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed. 1994)) .
  • an "isolated" construct or desired polypeptide is one which has been identified and separated and/or recovered from a component of the culture in which it is produced. Contaminant components of the culture are materials which would interfere with cell culture, diagnostic or therapeutic uses for the construct or desired polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes . In some embodiments, the construct or desired polypeptide will be purified to
  • Percent (%) amino acid sequence identity with respect to the polypeptide sequences referred to herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the expression "essentially free" with reference to a component of a cell culture medium means that the component is not deliberately added to or present in the culture medium, and if detectable is present as a result of unavoidable impurities in other components of the culture medium.
  • antibody includes all classes and subclasses of intact immunoglobulins, and also encompasses antibody fragments.
  • antibody specifically encompasses monoclonal antibodies, including antibody fragment clones .
  • the present invention may performed without any undue need for experimentation using, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following well-known publications, which are incorporated herein by reference:
  • the construct or fusion protein of the present invention comprises a peptide leader sequence containing or consisting essentially of LSTQ (SEQ ID NO: 19), and an amino sequence encoding a growth factor.
  • LSTQ SEQ ID NO: 19
  • the fusion protein of the present invention can be produced by recombinant DNA methodology.
  • a nucleic acid molecule encoding a leader peptide sequence of the present invention can be ligated to a nucleic acid molecule encoding a growth factor. Techniques for ligating such nucleic acids are well known in the art.
  • nucleic acid molecule can be further manipulated to introduce additional restriction enzyme cleavage sites, or to encode sites for enzymatic cleavage of the expressed protein.
  • nucleic acid of the present invention Once the nucleic acid of the present invention has been constructed it can be introduced into a vector for expression.
  • a "vector” is a plasmid or other DNA molecule which is capable of replicating autonomously within a host cell, and as such, is useful for performing two functions in conjunction with compatible host cells (a vector-host system) .
  • One function is to facilitate the cloning of the nucleic acid which encodes the construct or desired polypeptide of the invention, i.e., to produce usable quantities of the nucleic acid.
  • the other function is to direct the expression of the construct or desired polypeptide.
  • One or both of these functions is performed by the vector-host system.
  • the vectors will contain different components, depending upon the function they are to perform as well as the host cell with which they are to be used for cloning or expression.
  • the vector is a plasmid such as pGHXSC.4.
  • This plasmid has a modified RBS/spacer region and strategic 5 ' -codon alterations downstream from its powerful trc promoter (J. Brosius et al. J. Biol. Chem. 260, 3539, 1985; P. D. Vize & J. R. E. Wells, FEBS Lett. 213, 155, 1987) .
  • The- vector may be introduced into the host cell by methods which are also known in the art, for example by transformation, transduction or transfection techniques-.
  • the process may include the preliminary step of introducing coding for a cleavable bond between the first and second DNA sequences in the vector. This may involve subjecting the first DNA sequence to mutagenesis in order to introduce a cleavable sequence at the 3' end thereof.
  • mutagenesis methods are known in the art, and include in vitro PCR mutagenesis techniques.
  • a suitable in vitro mutagenesis PCR reaction may utilize an oligonucleotide which introduces a cleavable bond between the first and second DNA sequences.
  • a hydroxylamine-cleavable bond or a subtilisin-cleavable bond may be introduced.
  • an enzymatically-cleavable amino-acid motif may be introduced.
  • an amino acid motif which is susceptible to chemical cleavage may be introduced.
  • mutagenesis steps may be included to reduce the size of the sequence encoding the leader sequence and/or to introduce a restriction enzyme recognition site to facilitate additional mutagenesis steps.
  • the construct of the invention may be modified at the carboxy- or amino-terminal end, without loss of biological activity.
  • the present invention includes within its scope constructs which comprise further amino acids in addition to the "core" LSTQ residues of the leader sequence.
  • the construct may also include additional amino acids in the growth factor sequence, such as additions or modifications to this sequence .
  • constructs and desired polypeptides described herein may be produced by recombinant DNA techniques known to the person skilled in the art, for example those described in T. Maniatis et al . , in Molecular Cloning, A Laboratory Manual. CSH Lab. N. Y. (1989).
  • Production of the construct or polypeptide of the invention involves culturing a host cell which has been transformed with a vector according to the invention, and isolating the construct or polypeptide from the culture. Suitable methods for the expression and purification of IGF and analogues thereof are disclosed in US patent No. 5,330, 971. It is to be clearly understood that the present invention extends to biologically active fragments or functional analogues of human IGF, i.e.
  • IGF analogues or derivatives of human IGF in which the wild-type IGF sequence contains additions, deletions or substitutions by other amino acids or amino acid analogues, in which the biological activity of the IGF is retained.
  • IGF analogues suitable for use in the present invention include those described in US patents Nos . 5,077,276, 5,164,370,
  • analogues include des (1-3) IGF-I, des (1-6) IGF-II, analogues of IGF-I with amino acid substitutions at amino acid position 1 to 3, analogues of IGF-II with amino acid substitutions at amino acid position 1 to 6, porcine growth hormone-IGF fusion proteins and IGF analogues incorporating heparin-binding motifs which enable the IGF analogue to bind to fixed surfaces .
  • Methods for the identification, production and biological characterisation of active fragments, functional analogues or derivatives of growth factors are well known to those of ordinary skill in the art, and can be addressed with no more than routine experimentation.
  • the growth factor fragment, functional analogue or derivative has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, or 99% amino acid sequence homology, usually at least 70% amino acid sequence homology, preferably at least 90%, and even more preferably at least 95% amino acid sequence homology with the native growth factor amino acid sequence in question.
  • the biologically active fragment, functional analogue or derivative of the growth factor demonstrates biological activity.
  • the growth factor may have 100% amino acid sequence homology to the amino acid sequence of the corresponding human growth factor.
  • the IGF may have 100% amino acid sequence homology to the amino acid sequence of human IGF-I.
  • the amino acid sequence for human IGF-I is as follows: GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLE MYCAPLKPAKSA (SEQ ID NO: 1)
  • the IGF may have up to 100% amino acid sequence homology to the amino acid sequence of human IGF- II.
  • the amino acid sequence for human IGF-II is as follows:
  • porcine (Francis et al . (1989) J. Endocrinol. 122, 681-687), and murine (Stempien et al . (1986) DNA. 5, 357-361) sequences.
  • a suitable program for determining percentage sequence identity is BLAST 2.0 Sequence Comparison (NIH) .
  • NASH Sequence Comparison
  • the limiting parameters imposed for such a task are the default settings for the program. Therefore the person skilled in the art would readily, and with a reasonable expectation of success, be able to predict which alterations to the protein sequence would affect the structure of the growth factor, and affect its biological activity.
  • the alteration does not alter the growth factor domain responsible for affinity to the corresponding growth factor receptor.
  • polypeptide of the invention may be produced using methodology which is known to the person skilled in the art . Representative methods are described in the examples provided herein, but it will be clearly understood that other suitable methods are known in the art.
  • the leader sequence may be cleaved from the second amino acid sequence to release the relevant protein for subsequent use. Therefore the polypeptide is engineered to contain a chemical or enzymatic cleavage site.
  • conventional procedures are used.
  • construct or desired polypeptide may be expressed as an insoluble aggregate and/or is denatured, solubilization and/or renaturation may be effected using conventional techniques.
  • solubilization and/or renaturation may be effected using conventional techniques.
  • the construct or desired polypeptide may be folded and purified using a method such as :
  • the isolation of the constructs and desired polypeptides of the invention may be performed by any convenient method. Many suitable methods are known in the art. For example, the isolation of the polypeptide may be performed by ion-exchange chromatography, size exclusion chromatography, affinity chromatography or reverse phase high performance liquid chromatography.
  • the construct or desired polypeptide prior to isolation, is subjected to a dissolution and refolding step to release a biologically active product, especially where the host cell is a prokaryotic cell and the construct or desired polypeptide is expressed as an inclusion body.
  • the construct or desired polypeptide produced according to the methods of the invention may be isolated as inclusion bodies within the engineered unicellular organisms. In some circumstances the construct or desired polypeptide may be secreted rather than being expressed as an inclusion body.
  • the construct or desired polypeptide may be subjected to further processing steps as required.
  • the isolating step may comprise one or more of the following steps:
  • the construct or desired polypeptide may be subjected to a freeze-drying step.
  • the construct or desired polypeptide may also be sterilized, for example by microfiltration using an apparatus such as a Milli-GV filter unit (membrane area 4cm 2 ) .
  • the isolated product is cleaved so as to release the growth factor or desired polypeptide from the leader sequence.
  • the cleavage is performed using chemical or enzymatic cleavage.
  • the construct may be engineered to contain a chemical or enzymatic cleavage site, using methods known in the art, such as those described in US Patent No. 5,330,971.
  • the process may include the preliminary step of introducing a nucleic acid sequence which encodes a cleavable bond between the first and second DNA sequences in the vector.
  • the process may also include the preliminary steps of:
  • the digestion includes a subsequent purification step to provide a purified DNA fragment .
  • the second DNA sequence may be synthetic or recombinant, or may be obtained from a natural source.
  • the process may further include the preliminary step of chemically synthesising the second DNA sequence.
  • the chemical synthesis may be undertaken in any conventional manner.
  • the digestion and ligation steps may be undertaken in any conventional manner.
  • the process may include the preliminary steps of (a) providing a vector
  • the osmolality regulator maintains the medium at 200-350 mOsm.
  • the osmolality regulator maintains the medium at 200, 210, 220, 230, 240, 250, 260, 270 , 280 , 290 , 300 , 310 , 320 , 330 , 340 , or 350 mOsm .
  • the concentration of the energy source can be within the range of 1,000-10,000 mg/liter.
  • the concentration of the energy source is 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000,
  • the energy source is a monosaccharide .
  • the amino acids are selected from the group consisting of L-alanine, L-arginine, L- asparagine, L-aspartic acid, L-cystine, L-cysteine L- glutamic acid, glycine, L-histidine, L-isoleucine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L- proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-cysteine and L-valine.
  • the iron source is an inorganic ferric or ferrous salt which is provided in a concentration of from 0.25-5 mg/liter.
  • the iron source may be at a concentration of 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25,
  • recombinant iron sources include lactoferrin and transferrin or fragments thereof which are capable of sequestering iron and binding to the transferrin receptor.
  • organic iron sources include topoplone, ⁇ - thujaplacin, ⁇ -thujaplacin, ⁇ -thujaplacin, 3-hydroxypyran- 4-one, 3-hydroxy-2-methylpyran-4-one, 2-Ethyl-3- hydroxypyran4 -one , 3 , 5-dihydroxy-2-methylpyran-4-one, 2- benzyl-3 -hydroxypyran-4 -one, 2-ethyl-3 -hydroxy- 6- methylpyran-4-one and 2-hydroxy-1, 4 -naphthoquinone (Akers et al. J Bacteriol. 1980; 141(1): 164-168)
  • the medium may further comprise L-glutamine.
  • concentration of L-glutamine is within the range of 400-600 mg/liter.
  • concentration of L-glutamine is 400, 425, 450, 475, 500, 525, 550, 575, or 600 mg/liter.
  • the medium further comprises a lipid such as cholesterol, a steroid or a fatty acid in an amount of 0.05-10 mg/1.
  • a lipid such as cholesterol, a steroid or a fatty acid in an amount of 0.05-10 mg/1.
  • the concentration of the lipid factor is 0.05, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg/liter.
  • the pH of the medium can be maintained by use of a buffer at about 6.5 to about 7.5.
  • a buffer at about 6.5 to about 7.5.
  • the pH may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.
  • the medium may further comprise a peptide or protein digest, hydrolysate or extract.
  • the medium is essentially free of hypoxanthine and thymidine .
  • the medium may further comprise methotrexate . It has been found that the process described above produces biologically active growth factors such as IGF in high yield.
  • Quantification of the polypeptide on the basis of its specific molar absorbency may be performed by a calibrated RP-HPLC method, such as that described in Francis et al . (1992) J. MoI. Endo. (8) 213-223.
  • Determination of the potency of a construct in which the growth factor is IGF may comprise an assay which tests the stimulation of protein synthesis in rat L6 myoblasts, as described in G. L. Francis et al . Biochem. J. 233, 207- 213 (1986) , or by radioimmunoassay, using conventional methods. Suitable assays for other growth factors within the scope of the invention are well known in the art.
  • the construct of the invention may be administered by any suitable route, for example by parenteral injection. Although in critical care situations the preferred method of administration may be via addition to parenteral fluids, other methods may be appropriate.
  • the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.
  • the carrier or diluent, and other excipients will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case .
  • the dose rates and times for the administration of the construct to human subjects may for example be approximately 0.01 to 10 mg/kilogram/day. Dose rates of approximately 0.1 to 1 mg/kilogram/day are preferred.
  • the construct may be administered alone or with various diluents, carriers or excipients which have been chosen with respect to the intended method of administration and are well known.
  • Acceptable diluents, carriers or excipients are nontoxic at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids,- antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween (pol
  • fusion partner to prevent degradation and to increase expression in E. coll.
  • porcine growth hormone-derived fusion partner has been shown to promote the correct folding of the fusion protein into its preferred biologically active form (Milner et al . , (1995), Biochemical Journal 308:865- 871) .
  • a linker sequence which may be cleaved specifically can be inserted between the fusion partner and the desired protein, as is the case in the present invention.
  • the cleavage reaction may be achieved by either chemical means or by using a protease.
  • a protease for example the enzyme ⁇ -lytic protease from the soil bacterium Lysobacter enzymogenes is a good candidate for this role, following mutagenesis to contain the substitutions
  • the linkers selected in each case were, in order of preference, Asp-Ser-Thr-Met (DSTM) and Ile-Asn-Ala-His (INAH), for positions P4 , P3 , P2 and Pl, and, in keeping with the previously- established substrate preferences for mutant 9 at Pl.
  • DSTM Asp-Ser-Thr-Met
  • INAH Ile-Asn-Ala-His
  • mutant 9 has a preference of for cleavage at His or Met compared to GIn, as reported by Haggett et al . , (1994) (Archives of Biochemistry and Biophysics 314:132-141). Consequently our finding that our proposed linker motif, Leu-Ser-Thr-Gln
  • a chemically-synthesised nucleic acid encoding des (1-3) IGF-I was assembled in the plasmid pCR-Script.
  • the sequence was modified to introduce a nucleic acid sequence encoding the amino acid sequence LSTQ at a site 5' to the des (1-3) IGF-I gene. Mutagenesis was performed using the PCR technique.
  • the nucleotide sequence encoding the VNdes (1-3) IGF-I polypeptide with the encoded protein product is shown below: Hpal
  • oligonucleotide 29 base pairs long was used to introduce a HindiII (AAGCTT) recognition site 3' to the stop codons of the nucleotide sequence encoding the des(l- 3) IGF-I polypeptide.
  • the oligonucleotide sequence is shown below:
  • a second oligonucleotide 38 base pairs long was used to introduce the LSTQ sequence by mutagenesis.
  • the oligonucleotide sequence is shown below:
  • a mutant clone of pCR-Script comprising DNA encoding VNLSTQdes (1-3 ) IGF- I was selected, double stranded DNA was prepared, the DNA was digested with Hpal and Hindi II , and was subsequently isolated . This fragment was then cloned into the expression plasmid for MetpGH (1 -11) VNLSTQEGF from which the Hpal-Hindlll fragment (LSTQEGF) had been removed (pGHXSC.4) to give the expression plasmid for MetpGH(l- lUVNLSTQdes (1-3) IGF-I (see Example 4).
  • a mutant clone of pCR-Script comprising DNA encoding VNPAPMdes (1-3) IGF-I was selected and cloned into the Hpal- HindIIl treated MetpGH (1-11) VNLSTQEGF expression vector (pGHXSC.4) to provide the expression vector for MetpGH(l- 11) VNPAPMdes (1-3) IGF-I, using the method described in Example 2.
  • Example 4 Cloning PAPMpEGF 3' to the MetpQH (1-11) sequence in pGHXSC.4
  • a chemically-synthesised nucleic acid sequence encoding porcine EGF with codons optimised for E: coli expression contained 35 changes of 159 nucleotides of the native porcine EGF nucleotide sequence.
  • the nucleotide sequence encoding the PAPMpEGF polypeptide is shown below:
  • Mutagenesis was performed on the optimised pEGF sequence from Example 4, using PCR, in order to replace the nucleic acid encoding the amino acid sequence PAPM with nucleic acid encoding the amino acid sequence LSTQ 5' to the pEGF sequence.
  • An oligonucleotide 30 base pairs long was used to introduce a Hindi11 restriction site.3' to the stop codons of the nucleotide sequence.
  • the oligonucleotide sequence is shown below:
  • a mutant clone of pSTBlue-1 containing VNLSTQpEGF was selected and cloned into the expression vector
  • Cultures were grown in 3 litres of MinA medium at 37°C, pH 7.0, 60% pO2 and fed with glucose until an A600 of 8.0 was reached. Cultures were then induced with 200 mgs of isopropylthiogalactoside (IPTG) for 6 hrs and fed with an additional 50 g of glucose. Cells were homogenised at 9000 psi, and inclusion bodies were collected by centrifugation at 10,000 rpm at 4oC for 25 minutes. The inclusion bodies were washed by suspension in 30 mM NaCl, 10 mM KH 2 PO 4 , 0.5 mM ZnCl 2/ collected by centrifugation, and the wet paste stored at -80 0 C.
  • IPTG isopropylthiogalactoside
  • Inclusion bodies were dissolved in 8 M urea, 40 mM glycine, 1 mM EDTA, 0.1 M Tris, 20 mM DTT at pH 9.1 at a final concentration of 10 mg/ml, and incubated for 1 hour at room temperature.
  • a sample taken from the dissolved inclusion bodies was diluted 1:10 in 0.1% trifluoroacetic acid (TFA) and analysed by RP-HPLC. The results are shown in Table 1. This demonstrates that there was a 208% increase in yield when the leader sequence comprising LSTQ was used.
  • Example 6 The protein encoded by the nucleic sequence produced in Example 2 and produced as inclusion bodies as described in Example 6 did not require cleavage to achieve full biological activity equivalent to LONG 0 R 3 IGF-I (GroPep Ltd, Australia) after processing. This is demonstrated as follows.
  • Inclusion bodies were dissolved as described in Example 6, and refolded in 2 M urea, 10 mM glycine, 0.1 M Tris, 1.3 mM EDTA, 1 mM 2-hydroxymethyl disulphide and 0.4 mM DTT at pH 9.1 at room temperature for 90 minutes.
  • the protein concentration was 0.125 mg/ml .
  • Refolding was stopped by acidification with hydrochloric acid to pH 1.5, filtered, pumped on to a cation exchange column and eluted using an ammonium acetate gradient at pH 4.8.
  • the objective of this experiment was to summarise the evidence that modification of the des (1-3) IGF-I fusion protein leader sequence from GFAHY to LSTQ improved des CL3) IGF-I fusion protein expression levels in fermentations.
  • nucleotide sequence (SEQ ID NO: 50) coding for the region of the MetpGH (1-11) VNGFAHYdes (1-3) IGF-I polypeptide (SEQ ID NO: 51.) is shown below.
  • the numbering system is based on the expression vector.
  • the MetpGH(l-ll)VNLSTQdes (1-3) IGF-I construct was prepared in three steps. Firstly, the nucleotide sequence encoding VNPAPMdes (1-3) IGF-I from residue VaI12
  • the numbering system is based on the expression vector.
  • the MetpGH (1-11) VNGFAHYdes (1-3) IGF-I expression clone was fermented five times at the 14 L scale in a Chemap bioreactor (Bresatec Pty Ltd, Sydney) .
  • the fermentations were performed in minimal defined medium with a glucose feed.
  • the temperature was maintained at 37°C, the pH at 7.0, and dissolved oxygen at >50%.
  • the cultures typically reached an OD600 of 55 prior to induction with isopropylthiogalactoside (IPTG) and a termination OD600 of 75 at 5 hours after induction. Details of the parameters are described in King et al . 1992 J. MoI. Endocrinol. 8(1):29-41.
  • the MetpGH (1-11) VNLSTQdes (1-3) IGF-I expression clone was fermented at 3 L and 50 L-scales using either 3 L Applicon (Schiedam, the Netherlands) or 50 L Biostat (Sartorius BBI Systems GmbH, Melsungen, Germany) bioreactors .
  • the fermentations were performed in minimal defined medium with a glucose feed.
  • the temperature was maintained at 37 0 C and dissolved oxygen >50%.
  • the culture pH was 6.9 for the 3 L fermentation and 6.6 for the 50 L fermentation.
  • the cultures were induced with IPTG at an OD600 of 7-9 at the 3 L-scale and 30-35 at the 50L-scale.
  • the induction proceeded for 5 hours, yielding cultures with terminal OD600 values of 32-35 at the 3L-scale and 75-80 at the 50L-scale.
  • Inclusion bodies were recovered from the homogenised MetpGH (1-11) VNGFAHYdes (1-3) IGF-I culture broth using a continuous centrifuge. In the first pass, the centrifuge feed rate was approximately 290 mL/min. The recovered inclusion body paste was then washed by resuspension in a washing solution (final volume 2.5 L). The washed inclusion bodies were then recovered by a second centrifugation run with a feed rate of 650 mL/min.
  • Inclusion bodies were recovered from the homogenised 3L MetpGH (1-11) VNLSTQdes (1-3) IGF-I culture using a batch centrifuge (BeckmanCoulter, Australia) set to 9300 RPM. The centrifugation was performed at 4°C for 25 minutes.
  • Inclusion bodies were recovered from the homogenised 50 L MetpGH (1-11) VNLSTQdes (1-3) IGF-I culture broth using a continuous centrifuge (CEPA, Lahr, Germany) at 20,000 rpm. The homogenate was fed through the centrifuge at lL/min. Analysis of inclusion body purity and fermentation yield
  • the expression levels achieved during the fermentation of des (1-3) IGF-I fusion protein production clones were measured by dissolving known amounts of the respective inclusion body pastes (typically 0.3 g) in known volumes of reducing, denaturing buffer (typically 30 mL) in a 50 mL plastic tube.
  • the reducing denaturing buffer contained 8 M urea, 100 mM Tris, 40 mM glycine, 20 mM DTT with a pH of 9.0.
  • the fusion protein extractions proceeded for 60 minutes at room temperature while the reactions were mechanically mixed. After the extractions were complete, aliquots of the solubilized inclusion body extracts were removed and analysed by RP-HPLC.
  • Example 11 Enzymatic cleavage of the LSTQ linker sequence of the fusion protein to liberate des (1-3) IGF-I
  • the utility of the fusion protein linker sequence for specific enzymatic cleavage was investigated with partially purified MetpGH (1-11) VNLSTQdes (1-3) IGF-I fusion protein.
  • MetpGH (1-11) VNLSTQdes ( 1- 3) IGF-I fusion protein was extracted from inclusion bodies, refolded and purified by cation exchange chromatography as described in Example 8.
  • the eluate from the cation exchange chromatography column which contained the MetpGH (1-11) VNLSTQdes (1-3) IGF-I fusion protein was subsequently adjusted to 1.0mg/ml in a solution that contained 2.0 M urea and 0.1 M tris that had been adjusted to a pH of 8.0.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Endocrinology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés de production recombinante de protéines telles que des facteurs de croissance. En particulier, l'invention concerne des améliorations de procédés de production de protéines facteurs de croissance sous la forme de protéines de fusion. Dans un mode de réalisation, la protéine est un facteur de croissance tel que l'IGF ou l'EGF. La protéine de fusion comprend la séquence d'acides aminés LSTQ (SEQ ID N° 19) en tant que séquence leader servant de site de coupure pour une protéase alpha-lytique.
PCT/AU2006/001898 2005-12-15 2006-12-15 Production de protéines par voie recombinante Ceased WO2007068053A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US75035905P 2005-12-15 2005-12-15
US60/750,359 2005-12-15

Publications (1)

Publication Number Publication Date
WO2007068053A1 true WO2007068053A1 (fr) 2007-06-21

Family

ID=38162482

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2006/001898 Ceased WO2007068053A1 (fr) 2005-12-15 2006-12-15 Production de protéines par voie recombinante

Country Status (1)

Country Link
WO (1) WO2007068053A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009030720A3 (fr) * 2007-09-06 2009-04-30 Novozymes Biopharma Dk As Procédé pour produire une protéine recombinée
WO2010080494A1 (fr) * 2008-12-19 2010-07-15 Tercica, Inc. Procédés d'identification de produits de dégradation dans un échantillon polypeptidique.
CN101863982A (zh) * 2009-04-17 2010-10-20 哈药集团生物工程有限公司 一种用于升高血小板的融合蛋白及其制备方法
CN113582881A (zh) * 2021-07-29 2021-11-02 浙江新码生物医药有限公司 一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5330971A (en) * 1989-06-09 1994-07-19 Gropep Pty. Ltd. Growth hormone fusion proteins, methods of production, and methods of treatment
US20040023887A1 (en) * 1998-09-02 2004-02-05 Renuka Pillutla Insulin and IGF-1 receptor agonists and antagonists

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5330971A (en) * 1989-06-09 1994-07-19 Gropep Pty. Ltd. Growth hormone fusion proteins, methods of production, and methods of treatment
US20040023887A1 (en) * 1998-09-02 2004-02-05 Renuka Pillutla Insulin and IGF-1 receptor agonists and antagonists

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIEN S. ET AL.: "Isolating Substrate for an Engineered alpha-Lytic Protease", JOURNAL OF PROTEIN CHEMISTRY, vol. 22, no. 2, February 2003 (2003-02-01), pages 155 - 166, XP019284280 *
LIEN S. ET AL.: "Linkers for Improved Cleavage of Fusion Protein with an Engineered alpha-Lytic Protease", BIOTECHNOLOGY AND BIOENGINEERING, vol. 74, no. 4, 20 August 2001 (2001-08-20), pages 335 - 343, XP003014121 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009030720A3 (fr) * 2007-09-06 2009-04-30 Novozymes Biopharma Dk As Procédé pour produire une protéine recombinée
WO2010080494A1 (fr) * 2008-12-19 2010-07-15 Tercica, Inc. Procédés d'identification de produits de dégradation dans un échantillon polypeptidique.
CN101863982A (zh) * 2009-04-17 2010-10-20 哈药集团生物工程有限公司 一种用于升高血小板的融合蛋白及其制备方法
CN113582881A (zh) * 2021-07-29 2021-11-02 浙江新码生物医药有限公司 一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物

Similar Documents

Publication Publication Date Title
JP5634971B2 (ja) 培地へのペプチドの直接発現
US6037143A (en) Enzymatic method for modification of recombinant polypeptides
Shin et al. High-level production of human growth hormone in Escherichia coli by a simple recombinant process
CA2237296C (fr) Procede de preparation de peptides par l'intermediaire de proteines de fusion issues de la streptavidine
CN100587073C (zh) 人成纤维细胞生长因子-21的重组表达
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
JP4857279B2 (ja) カルボキシ末端をアミド化したペプチドの製造方法
Liew et al. Preparation of recombinant thioredoxin fused N-terminal proCNP: analysis of enterokinase cleavage products reveals new enterokinase cleavage sites
CN101186940A (zh) 获得具有正确键合的胱氨酸键的胰岛素或胰岛素衍生物的方法
JP4624495B2 (ja) ヒト・インスリンの生成
CN110128521A (zh) 用于生产重组融合蛋白的辅助蛋白、编码基因、重组融合蛋白、重组表达载体及制备方法
US9096843B2 (en) Peptidyl α-hydroxyglycine α-amidating lyases
US6103882A (en) Method of producing a 19P2 ligand
Ray et al. Production of salmon calcitonin by direct expression of a glycine-extended precursor in Escherichia coli
WO2010062279A1 (fr) Procédé de production d’insuline humaine recombinante
Liu et al. Large scale preparation of recombinant human parathyroid hormone 1–84 from Escherichia coli
CN112500495A (zh) 一种elp-ⅲ型胶原蛋白的纯化方法及应用
WO2007068053A1 (fr) Production de protéines par voie recombinante
WO2007112676A1 (fr) Protéine hybride de l'hormone parathyroïde humaine 1-34 et vecteurs d'expression correspondants
Mahmoud et al. Production of a biologically active novel goldfish growth hormone in Escherichia coli
Wilkinson et al. Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II
Mayne et al. Direct expression of human growth in Escherichia coli with the lipoprotein promoter
WO1986003779A1 (fr) Vecteur de secretion de polypeptides, micro-organismes transformes par ledit vecteur, et procede de preparation de polypeptides avec lesdits micro-organismes
NZ232255A (en) Glucagon of high purity and production using a fusion protein
KR100235315B1 (ko) 융합단백질을 이용한 인성장호르몬의 제조방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06828010

Country of ref document: EP

Kind code of ref document: A1