[go: up one dir, main page]

WO2005024012A1 - Expression accrue d'un polypeptide modifie - Google Patents

Expression accrue d'un polypeptide modifie Download PDF

Info

Publication number
WO2005024012A1
WO2005024012A1 PCT/DK2004/000597 DK2004000597W WO2005024012A1 WO 2005024012 A1 WO2005024012 A1 WO 2005024012A1 DK 2004000597 W DK2004000597 W DK 2004000597W WO 2005024012 A1 WO2005024012 A1 WO 2005024012A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
modified
host cell
codon
expression
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/DK2004/000597
Other languages
English (en)
Inventor
Jesper Vind
Shamkant Anant Patkar
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 AS
Original Assignee
Novozymes AS
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 Novozymes AS filed Critical Novozymes AS
Publication of WO2005024012A1 publication Critical patent/WO2005024012A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the p resent i nvention relates to a method for preparing a n ucleotide sequence e ncoding a modified polypeptide and a method for preparing a modified polypeptide, wherein said methods results in increased expression of the modified polypeptide as compared to expression of a parent polypeptide.
  • the present invention also relates to a method of screening a library of modified nucleotide sequences for nucleotide sequences having an increased expression compared to expression of a parent nucleotide sequence.
  • disulfide bonds which is a covalent bond formed between the sulphur g roups of two cysteine residues in the protein. Formation of disulfide bonds in proteins occur both co- and post-translationally.
  • Disulfide-bond chemistry has been applied to study protein folding, structure and stability as reviewed by WJ Wedemeyer et al. (Biochemistry (2000), 39 (15), pp. 4207-4216). The roles of disulfide bonds in structure, conformational stability and catalytic activity of phosphorlipase A2 has also been investigated (H Zhu et al., Biochemistry (1995), 34, pp. 15307-15314).
  • DP Humphreys et al. (FEBS Letter (1996) 380, pp. 194-197) describes that co-expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab' fragment expressed in Escherichia coli.
  • PDI human protein disulphide isomerase
  • the p resent i nvention relates to a method for preparing a n ucleotide sequence e ncoding a modified polypeptide comprising: a) providing a nucleotide sequence encoding a parent polypeptide wherein said parent polypeptide comprises at least two Cys residues forming a disulfide bond; b) modifying at least one codon of the nucleotide sequence, wherein said codon specifies a Cys residue involved in a disulfide bond, so that the modified codon does not specify a Cys residue; and
  • the present invention also relates to a method for screening a library of modified nucleotide sequences, comprising: a) providing a n ucleotide s equence e ncoding a p arent p olypeptide w herein s aid p arent polypeptide comprises at least two Cys residues forming a disulfide bond; b) generating a library of modified nucleotide sequences by modifying at least one codon of the nucleotide sequence provided in step a), wherein said codon specifies a Cys residue involved in a disulfide bond, so that the modified codon does not specify a Cys residue; c) selecting a modified nucleotide sequence which has increased expression as compared to expression of a parent nucleotide sequence.
  • the present invention also relates to a method for preparing a modified polypeptide, comprising: a) cultivating a host cell under conditions suitable for expression of a modified polypeptide, wherein the host cell comprises a nucleotide sequence which has been modified by modifying at least one codon specifying a Cys residue involved in a disulfide bond in a parent polypeptide so that said codon does not specify a Cys residue, and wherein said modification results in increased expression of the modified polypeptide as compared to expression of the parent polypeptide; b) recovering the modified polypeptide from the cultivation medium.
  • parent polypeptide is in the context of the present invention to be understood as a polypeptide which is modified to create a "modified polypeptide".
  • the parent polypeptide may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means.
  • the parent polypeptide may be a variant of a naturally occurring polypeptide which has been modified by s ubstitution, d eletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide.
  • variant is in this context to be understood as a polypeptide which has been modified compared to a parent polypeptide at one or more amino acid positions.
  • modification(s) or “modified” is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein.
  • the modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
  • modified polypeptide is in the context of the present invention to be understood as a variant of a parent polypeptide which has been modified by a method of the present invention.
  • heterologous is in the context of the present invention to be understood as being derived from a different origin.
  • heterologous expression refers to expression of a polypeptide in a host cell, wherein said polypeptide is not expressed by the host cell in nature.
  • homologous is in the context of the present invention to be understood as being derived from the same origin.
  • homologous expression refers to expression of a polypeptide in a host cell, wherein said polypeptide is expressed by the host cell in nature.
  • disulfide bond or "disulfide bridge” is in the context of the present invention to be understood as a covalent bond between the sulphur atoms of two Cysteine residues in a polypeptide. Two such Cysteine residues linked by a disulfide bond may be designated as a cystine residue (see e.g. Creighton TE (1993), Proteins; Structures and Molecular Properties, 2nd Edition W.H: Freeman and Company, p. 18).
  • the term "increased expression” is in the context of the present invention to be understood as the amount of modified polypeptide expressed by a given number of host cells and within a given period of time is higher than the amount of a parent polypeptide expressed by the same number of host cells and within the same period of time and where expression of the modified and the parent polypeptide is performed under the same conditions.
  • “same conditions” refer to that the nucleotide sequences encoding the modified and the parent polypeptide are introduced by the same means into the same host cell, the host cell is cultured under the same conditions and the amount of polypeptides are measured by the same means.
  • polypeptide is in the context of the present invention intended to encompass peptides, polypeptides and proteins.
  • nucleotide sequence refers in the context of the present invention to the order of nucleotides in a nucleic acid, which may be DNA or RNA.
  • codon refers to the triplet of nucleotides specifying/encoding an amino acid or designating a signal for start or stop of translation.
  • synthetic genes are used more often to express proteins the origin of a synthetic gene is in the context of the present invention to be understood as referring to the origin of the gene which is used as a model for the synthetic gene. The same is to be understood for synthetic proteins.
  • an insertions and/or a substitution of amino acid(s) the following nomenclature is used in the present invention: original amino acid(s), position(s), deleted/inserted/substituted amino acid(s).
  • substitution of Glutamic acid for glycine in position 195 is designated as: Gly 195 Glu or G195E
  • a deletion of glycine in the same position is: Gly 195 * or G195 *
  • insertion of an additional amino acid residue such as lysine is: Gly 195 GlyLys or G195GK
  • an insertion in such a position is indicated as: * 36 Asp or * 36D for insertion of an aspartic acid in position 36
  • Multiple mutations are separated by pluses, i.e.: Arg 170 Tyr + Gly 195 Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glut
  • the inventors of the present invention have found that expression of a polypeptide may be increased by disrupting one or more disulfide bond of the polypeptide, i.e. decreasing the number of disulfide bonds in a parent polypeptide, and thereby creating a modified polypeptide, increases expression of the modified polypeptide compared to the parent polypeptide.
  • the number of disulfide bonds in the modified polypeptide of the present invention may be at least 1 , such as at least 2 or 3 or 4 or 5 or 6 or 7 or 8 less than the number of disulfide bonds in the parent polypeptide, i.e.
  • the number of Cysteine (Cys) residues in the modified polypeptide may be at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 less than the number of Cys residues in the parent polypeptide.
  • some disulfide bond(s) in a polypeptide may also be important for other characteristics than the level of expression, such as the stability, structure and/or function of the polypeptide, it may be an advantage if not all of the disulfide bonds of the parent polypeptide are modified.
  • the modified polypeptide comprises at least 1 , such as at least 2 or 3 or 4 or 5 or 6 or 7 or 8 disulfide bonds.
  • the number of disulfide bonds in a p arent p olypeptide m ay vary for d ifferent p olypeptides a nd t he p ercentage of i ts d isulfide bonds which are important for other characteristics than expression may also vary for different polypeptides.
  • the number of disulfide bonds and/or Cys residues in the modified polypeptide of the present invention is between 0- 10% or 0-20% or 0-30% or 0-40% or 0-50% or 0-60% or 0-70% or 0-80% or 0-90% or 5-25% or 5-50% or 5-75% or 5-95% or 20-60% or 40-90% of the number of disulfide bonds or Cys residues, respectively in the parent polypeptide.
  • the modified polypeptide may comprise at least 2, such as at least 4 or at least 6 or at least 8 or at least 10 or at least 12 or at least 14 or at least 16 or at least 18 fewer Cys residues than the parent polypeptide.
  • the function of the modified polypeptide is the same as the function of the parent polypeptide.
  • the activity of the modified polypeptide may be at least 100%, such as at least 95% or at least 90% or at least 80% or at least 70%) or at least 60% or at least 50% or at least 40% or at least 30% or at least 20% or at least 10%) of the activity of the parent polypeptide. It is also envisioned that modification of the parent polypeptide may affect the activity of the modified polypeptide positively, so that the activity of the modified polypeptide is higher than the activity of the parent polypeptide. Thus the activity of the modified polypeptide may be at least 110%, such as at least 120% or at least 130% or at least 150% or at least 175% or at least 200% of the activity of the parent polypeptide.
  • the term "activity" refers to the function of the parent polypeptide, e.g. if the parent polypeptide is an enzyme it refers to the catalytic activity of said enzyme, if the parent polypeptide is an antibody or antigen it refers to the binding affinity of said compounds to an antigen/antibody, respectively.
  • S imilarly i f t he p arent p olypeptide i s a r eceptor o r r eceptor-binding-molecule i t may refer to the binding affinity of said compounds to a receptor-binding-molecule/receptor, respectively.
  • Assays for testing the function of individual polypeptides are well-known to a person skilled in the art. Many assays for detecting enzymatic activity are based on the conversion of a substrate to a product.
  • Enzymatic activity of polypeptides may be determined by using different methods such as spectrophotometry, fluorescence changes, thermal changes, refractive-index changes, paper- and Thin-layer chromatography, electrochemical methods or radiometric methods using radioactive substrates, or by titration with an acid or a base. Examples of these methods are e.g. described in "Enzyme Assays: A Practical approach", Edited by R. Eisenthal and M.J.Danson in The practical Approach series, Editors: D.Rickwood and B.D. Hames IRL Press at Oxford University Press Oxford New York, Tokyo1993.
  • binding may be detected with immunochemical or radiometric methods, such as precipitation of a complex comprising labelled ligands or labelled antigens.
  • Immunoprecipitation, Radio-lmmuno assay or Enzyme labelled Immuno assays may also be used for showing binding of ligands to receptors or binding of antigens to antibodies. Examples of these methods are described in "Laboratory techniques in Biochemistry and molecular biology", general editors: R.H.Burdon and P.H.van Knippenberg Volume 15 and "Practice and theory of enzyme immunoassays" by P.Tijssen, Elsevier publication 1987.
  • the parent polypeptide may be any polypeptide comprising at least one disulfide bond. Without being bound to any particular theory the inventors of the present invention believe that one reason for low expression of polypeptides in recombinant host cells may be that the formation of disulfide bonds in polypeptides is a rate-limiting step in the process of expression. Hence the present invention may in particular be useful for modification of parent polypeptides which comprise a relative h igh n umber of d isulfide bonds. T hus the method of the p resent invention may be particularly useful for a parent polypeptide comprising more than one disulfide bond, such as at least 2 disulfide bonds, e.g. at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 disulfide bonds.
  • the parent polypeptide may have any function, e.g. it may be an enzyme, an antimicrobial peptide, an antibody, a receptor, a hormone or a transport protein, such as haemoglobin.
  • the parent polypeptide is an enzyme.
  • the enzyme may belong to a known class of enzymes, or it may be of an unknown enzyme class, e.g. an enzyme having a desired functional activity but not necessarily belonging to a known enzyme class.
  • enzyme class E.C. refers to the internationally recognized enzyme classification system, Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press, Inc., 1992.
  • the enzyme may belong to one of the following classes: oxidoreductases (EC 1.-.- .-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).
  • oxidoreductases EC 1.-.- .-
  • transferases EC 2.-.-.-
  • hydrolases EC 3.-.-.-
  • lyases EC 4.-.-.-
  • isomerases EC 5.-.-.-
  • ligases EC 6.-.-.-.-
  • oxidoreductases include peroxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4)], while transferases may be transferases belonging to any of the following sub-classes:
  • the transferase may be a transglutaminase (protein-glutamine gamma- glutamyltransferase; EC 2.3.2.13).
  • hydrolases examples include: Carboxylic ester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3); phytases (EC 3.1.3.-), e.g. 3-phytases (EC 3.1.3.8) a nd 6-phytases ( EC 3.1.3.26); glycosidases (EC 3.2, which fall within a group denoted herein as "carbohydrases”), such as alpha-amylases (EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases.
  • Carboxylic ester hydrolases EC 3.1.1.-
  • phytases EC 3.1.3.-
  • 3-phytases e.g. 3-phytases (EC 3.1.3.8) a nd 6-phytases ( EC 3.1.3.26)
  • glycosidases EC 3.2, which fall within a group denoted herein as "carbohydr
  • hydrolases include xyloglucanase, arabinase, rhamno- galactoronase, pectinases, ligninases (for example polyphenol hydrolase).
  • the parent polypeptide may be a phospholipase, such as phosphorlipase A2 (E.C.3.1.1.4), e.g. it may be phospholipase A2 from pig or a variant thereof such as phospholipase A2 from pig with a S21 R mutation, i.e. the polypeptide given by SEQ ID NO.1.
  • carbohydrase is used to denote not only enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five- and six-membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six-membered ring structures such as D-glucose to five-membered ring structures such as D-fructose.
  • Carbohydrases of relevance include the following (EC numbers in parentheses): alpha-amylases (3.2.1.1), beta-amylases (3.2.1.2), glucan 1 ,4-alpha-glucosidases (3.2.1.3), cellulases (3.2.1.4), endo-1 ,3(4)-beta-glucanases (3.2.1.6), endo-1 ,4-beta-xylanases (3.2.1.8), dextranases (3.2.1.11), chitinases (3.2.1.14), polygalacturonases (3.2.1.15), lysozymes (3.2.1.17), beta-glucosidases (3.2.1.21), alpha-galactosidases (3.2.1.22), beta-galactosidases (3.2.1.23), mannanase (3.2.1.25), amylo-1 ,6-glucosidases (3.2.1.33), xylan 1 ,
  • the parent polypeptide may be an antimicrobial polypeptide, wherein the term "antimicrobial” is intended to mean that there is a bactericidal (capable of killing bacterial cells) and/or a bacteriostatic (capable of inhibiting bacterial proliferation) and/or fungicidal (capable of killing fungal cells) and/or fungistatic (capable of inhibiting fungal proliferation) effect and/or a virucidal (capable of inactivating virus) effect.
  • it may be an antimicrobial polypeptide of 2-100 amino acids.
  • the parent polypeptide may be a cystine-rich antimicrobial polypeptide, such as alpha-Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3; beta-Defensin-12, Drosomycin, gammal- purothionin, and Insect defensin A.
  • alpha-Defensin HNP-1 human neutrophil peptide
  • HNP-2 and HNP-3 human neutrophil peptide
  • beta-Defensin-12 beta-Defensin-12
  • Drosomycin gammal- purothionin
  • Insect defensin A Insect defensin A.
  • a suitable parent polypeptide is Plectasin, which is an antimicrobial peptide derived from Pseudoplectania nigrella (SEQ ID NO:2 in PA 2001 01732).
  • Another example of a suitable parent polypeptide includes the antimicrobial polypeptide AFP derived from Aspergillus giganteus which comprises 8 Cysteines.
  • the parent polypeptide may be an antibody, e.g. a monoclonal or a polyclonal antibody.
  • the parent polypeptide may be a polypeptide isolated from a natural source, i.e. a wild-type polypeptide, or it may be a polypeptide isolated from a natural source in which subsequent modifications h ave b een m ade. F or i nstance, the p arent p olypeptide m ay b e a variant of a naturally occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide.
  • the parent polypeptide may be a polypeptide which has been prepared by a DNA shuffling technique, such as described by J.E. Ness et al., Nature Biotechnology, 17, 893-896 (1999). Further, a parent polypeptide may be constructed by standard techniques for artificial creation of diversity, such as described in WO 95/22625 or Stemmer WPC, Nature 370:389-91 (1994).
  • the parent polypeptide may be derived from any organism or it may be an artificial polypeptide.
  • the parent polypeptide may be derived from a eukaryotic cell, e.g. a mammalian cell, a fungal cell, an insect cell, a plant cell or an amphibian cell or it may be derived from a prokaryotic cell, such as a bacterium, e.g. a gram-negative or a gram-positive bacterium.
  • a bacterium e.g. a gram-negative or a gram-positive bacterium.
  • Examples of eukaryotic cells or prokaryotic cells from which the parent polypeptide may be derived include those described as host cells below.
  • the present invention provides a method for preparing a nucleotide sequence encoding a modified polypeptide, a method for screening a library of modified nucleotide sequences and a method for preparing a modified polypeptide.
  • Each of said methods are based on the finding that expression of a polypeptide in a host cell is increased by modifying at least one codon of the nucleotide sequence encoding a parent polypeptide, wherein said codon specifies a Cys residue involved in a disulfide bond in the parent polypeptide and wherein the modified codon does not specify a Cys residue.
  • Modification of the codon specifying a Cys residue involved in disulfide bond may in particular be performed so as to delete or substitute the Cys residue, i.e. the codon may be modified by deletion of the codon or by substitution of one or more nucleotides of the codon so that said modified codon no longer specifies a Cys residue. It may be an advantage if the m odified codon does not designate termination of translation, i.e. a stop codon.
  • the codon may be modified so as to substitute the Cys residue involved in a disulfide bond with a different amino acid.
  • the codon may be modified so as to substitute the Cys residue with an amino acid which introduces few overall and/or local changes besides disruption of the disulfide bond in the parent polypeptide.
  • the codon may be modified so as to substitute the Cys residue with an Ala residue, a Gly residue or a Ser residue.
  • nucleotide sequence Methods for deleting and/or substituting nucleotides in a nucleotide sequence are well known to a person skilled in the art and may include methods like site-directed mutagenesis or PCR generated mutagenesis see e .g. "Molecular cloning: A laboratory manual” (Sambrook et a I. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.)) or "Current protocols in Molecular Biology” (John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.)).
  • different parent polypeptides may generally comprise a different number of disulfide bonds and/or it may also be a different percentage of those that are important for other characteristics than the level of expression of said polypeptide.
  • the number of disulfide bonds which is necessary to disrupt to increase expression of a given polypeptide may be different for different parent polypeptides.
  • 1-17 or 5-17 or 9-17 or 13-17, or at least 18; e.g. 1-18 or 6-18 or 12-18 codons specifying a Cys involved in a disulfide bond in the parent polypeptide may be modified. More particularly at least two codons specifying both of the Cys residues forming a disulfide bond may be modified, i.e. both of the two codons which specify a cystine are modified.
  • the host cell of the present invention may be any cell capable of being genetically manipulated to express a polypeptide.
  • the host cell may be a heterologous host cell, i .e. a host cell which does n ot e xpress t he parent polypeptide in nature or it may be a homologous host cell, i.e. a host cell which expresses the parent polypeptide in nature.
  • the host cell may be a eukaryotic cell, e.g. a mammalian cell, a fungal cell, an insect cell, a plant cell or an amphibian cell. Probably due to characteristics such as glycosylation and/or folding it is often preferable to express eukaryotic polypeptides in eukaryotic host cells and to express prokaryotic polypeptides in prokaryotic host cells.
  • the host cell may be a eukaryotic cell if the parent polypeptide is a eukaryotic polypeptide or the host cell may be a prokaryotic host cell if the parent polypeptide is a prokaryotic polypeptide.
  • eukaryotic polypeptide a nd " prokaryotic p olypeptide” refer to the origin of the parent polypeptide, i.e. whether it originally derives from a eukaryotic or prokaryotic cell.
  • Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
  • Examples of insect cells include a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214).
  • the host cell is a fungal cell.
  • Fungi as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
  • Examples of Basidiomycota include mushrooms, rusts, and smuts.
  • Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.
  • Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya.
  • mitosporic fungi examples include Aspergillus, Penicillium, Candida, and Alternaria.
  • Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.
  • the fungal host cell is a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae.
  • the latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Pichia, Kluyveromyces and Saccharomyces).
  • the basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidioholus, Filobasidium, and Filobasidiella.
  • yeasts belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sorobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980.
  • yeast and manipulation of yeast genetics are well known in the art (see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A.O.M., editors, 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., editors, 2nd edition, 1987; and The Molecular Biology of the Yeast Saccharomyces, Strathern et al., editors, 1981).
  • the yeast host cell is a cell of a species of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia.
  • the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
  • the yeast host cell is a Kluyveromyces lactis cell.
  • the yeast host cell is a Yarrowia lipolytica cell.
  • the fungal host cell is a filamentous fungal cell.
  • filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).
  • the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.
  • the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell.
  • the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell.
  • the filamentous fungal host cell is a Fusarium venenatum (Nirenberg sp. nov.) cell.
  • the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
  • suitable bacterial host cells include gram positive bacteria of the genus Bacillus such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus megaterium, Bacillus circulans, and Bacillus lautus and of the genus Streptomyces such as Streptomyces lividans.
  • suitable gram-negative bacteria comprise bacteria of the genus Escherichia such as E. coli.
  • the transformation of the bacterial host cell may for instance be effected by protoplast transformation or by using competent cells in a manner known per se.
  • Another suitable bacterial cell is a cell of a Pseudomonas spp. such as Pseudomonas cepacia, Pseudomonas fragi, Pseudomonas gladioli, Pseudomonas fluorescens, Pseudomonas stutzeri, Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas glumae or Pseudomonas aeruginosa. Expression of polypeptides in a host cell
  • Expression of a polypeptide i n a h ost cell i n cludes i ntroduction of the n ucleotide sequence encoding said polypeptide into the host cell to produce a recombinant host cell and subsequently culturing the recombinant host cell under conditions suitable for transcription and translation of said nucleotide sequence into a polypeptide sequence.
  • this usually also include secretion of said polypeptide.
  • One method of the present invention relates to screening a library of modified nucleotide sequences.
  • Said library of modified nucleotide sequences is generated by modifying at least one codon of a nucleotide sequence encoding a parent polypeptide, wherein said codon specifies a Cys residue i nvolved in a d isulfide b ond i n t he p arent p olypeptide, s o t hat s aid codon does not specify a Cys residue.
  • Methods for generating a library of modified nucleotide sequences from a nucleotide sequence encoding a parent polypeptide may be performed by any means.
  • procedures for extracting DNA from a cellular nucleotide source and preparing a gene library are described in e.g. Pitcher et al. (1989), Dretzen, G. et al. (1981), WO 94/19454,3969, Diderichsen et al. (1990).
  • Procedures for preparing a gene library from an in vitro made synthetic nucleotide source can be found in (e.g. described by Stemmer, (1994) or WO 95/17413).
  • Error prone PCR employs a low fidelity replication step to introduce random point mutations at each round of amplification (Caldwell and Joyce (1992), PCR Methods and Applications vol.2 (1), pp.28-33).
  • Error-prone PCR mutagenesis is performed using a plasmid encoding the wild-type, i.e. wt, gene of interest as template to amplify this gene with flanking primers under PCR conditions where increased error rates leads to introduction of random point mutations.
  • the PCR conditions utilized are typically: 10 mM Tris-HCI, pH 8.3, 50 mM KCI, 4 mM MgCI2, 0.3 mM MnCI2, 0.1 mM dGTP/dATP, 0.5 mM dTTP/dCTP, and 2.5 u Taq polymerase per 100 micro L of reaction.
  • the resultant PCR fragment is purified on a gel and cloned using standard molecular biology techniques.
  • Oligonucleotide directed mutagenesis in single codon position e.g. by SOE-PCR is described by Kirchhoff and Desrosiers, PCR Methods and Applications, 1993, 2, 301-304.
  • This method is performed as follows: Two independent PCR reactions are performed with 2 internal, overlapping primers, wherein one or both contain a mutant sequence and 2 external primers, which may encode restriction sites, thereby creating 2 overlapping PCR fragments. These PCR fragments are purified, diluted, and mixed in molar ratio 1 :1. The full length PCR product is subsequently obtained by PCR amplification with the external primers. The PCR fragment is purified on gel and cloned using standard molecular biology techniques. (3) Oligonucleotide directed randomization in single codon position, such as saturation mutagenesis, may be done e.g. by SOE-PCR as described above, but using primers with randomized nucleotides.
  • NN(G/T) wherein N is any of the 4 bases G,A,T or C, will yield a mixture of codons encoding all possible amino acids.
  • Combinatorial site-directed mutagenesis libraries may be employed, where several codons can be mutated at once using (2) and (3) above. For multiple sites, several overlapping PCR fragments are assembled simultaneously in a SOE-PCR setup.
  • Another protocol employs synthetic gene libraries preparation. Wild type, i.e. wt, genes can be assembled from multiple overlapping oligonucleotides (typically 40-100 nucleotides in length; (Stemmer et al., (1995), Gene 164, 49-53).
  • the resulting assembled gene will contain mutations at various positions with mutagenic rates corresponding to the ratios of wt to mutant primers.
  • Still another method employs multiple mutagenic primers to generate libraries with multiple mutated positions.
  • an uracil-containing nucleotide template encoding a polypeptide of interest is generated and 2-50 mutagenic primers corresponding to at least one region of identity in the nucleotide template are synthezised so that each mutagenic primer comprises at least one substitution of the template sequence (or: insertion/deletion of bases) resulting in at least one amino acid substitution (or insertion/deletion) of the amino acid sequence encoded by the uracil-containing nucleotide template.
  • the mutagenic primers are then contacted with the uracil-containing nucleotide template under conditions wherein a mutagenic primer anneals to the template sequence.
  • Libraries may be created by shuffling e.g. by recombination of two or more wt genes or genes encoding variant proteins created by any combination of methods (1)-(6)
  • Introducing a library of modified nucleotide sequences into a host cell, expressing the modified nucleotide sequences in a host cell, isolation or purification of the expressed modified polypeptide may be performed similarly as for any nucleotide sequence. Introducing a nucleotide sequence into a host cell
  • a nucleotide sequence encoding a polypeptide is generally introduced into a host by cloning said sequence into an expression vector and subsequently introducing said expression vector into a host cell.
  • the choice of expression vector will often depend on the host cell into which it is to be introduced.
  • a suitable vector include a linear or closed circular plasmid or a virus.
  • 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, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, PACYC177, pACYC184, pUB110, pE194, pTA1060, and pAM ⁇ l.
  • Examples of origin of replications for use in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4, and the combination of CEN3 and ARS1.
  • the origin of replication may be one having a mutation which makes it function as temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • Vectors which are integrated into the genome of the host cell may contain any nucleic acid sequence enabling integration into the genome; in particular it may contain nucleic acid sequences facilitating integration into the genome by homologous or non-homologous recombination.
  • the vector system may be a single vector, e.g. plasmid or virus, or two or more vectors, e .g. p lasmids or virus', which together contain the total n ucleotide sequence to be introduced into the genome of the host cell, or a transposon.
  • the vector may in particular be an expression vector in which the nucleotide sequence encoding the modified polypeptide or the parent polypeptide of the invention is operably linked to additional segments or control sequences required for transcription of the DNA.
  • operably linked indicates that the segments are arranged so that they function in concert for their i ntended p urposes, e .g. t ranscription i nitiates i n a p romoter a nd p roceeds t hrough the nucleotide sequence encoding the modified polypeptide or the parent polypeptide.
  • control sequences include a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal sequence and a transcription terminator.
  • control sequences include a promoter and transcriptional and translational stop signals.
  • the promoter may be any nucleotide sequence that 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.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus subtilis levansucrase gene (sacB), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus subtilis alkaline protease gene, or the Bacillus pumilus xylosidase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731).
  • sacB Bacillus subtilis le
  • phage Lambda PR or PL promoters examples include the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters or the Streptomyces coelicolor agarase gene (dagA). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.
  • promoters for use in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (as described in U.S.
  • promoters for use in filamentous fungal host cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral (alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and glaA promoters.
  • TAKA amylase a hybrid of the promoters from the genes encoding Aspergillus niger neutral (alpha-amylase and Aspergillus oryzae triose phosphate isomerase
  • glaA promoters glaA promoters.
  • yeast host cells examples include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen.
  • yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.
  • useful promoters include viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • viral promoters such as those from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus, and bovine papilloma virus (BPV).
  • suitable promoters for use in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa califomica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
  • the polyhedrin promoter US 4,745,051; Vasuvedan et al., FEBS Lett. 311 , (1992) 7 - 11
  • the P10 promoter J.M. Vlak et al., J. Gen. Virology 69, 1988, pp. 76
  • the nucleotide sequence encoding the modified polypeptide or the parent polypeptide of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, neomycin, hygromycin, methotrexate, or resistance to heavy metals, virus or herbicides, or which provides for prototrophy or auxotrophs.
  • bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, resistance.
  • a frequently used mammalian marker is the dihydrofolate reductase gene (DHFR).
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • a selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltrans- ferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'- phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as well as equivalents from other species.
  • amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae are the amdS and pyrG markers of Aspergillus nidulans or Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.
  • selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the nucleotide sequence encoding the polypeptide in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the polypeptide.
  • the secretory signal sequence may be that normally associated with the modified polypeptide or parent polypeptide of the present invention or it may be from a gene encoding another secreted protein.
  • nucleotide sequences coding for the modified polypeptide or the parent polypeptide of the present invention are well known to persons skilled in the art (cf., for instance, Sambrook et al.). More than one copy of a nucleotide sequence encoding a modified polypeptide or a parent polypeptide of the present invention may be inserted into the host cell to amplify expression of the nucleotide sequence.
  • Stable amplification of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome using methods well known in the art and selecting for transfectants/transformants.
  • the nucleotide sequence constructs of the present invention may also comprise one or more nucleotide sequences which encode one or more factors that are advantageous in the expression of the modified polypeptide or the parent polypeptide, e.g., an activator (e.g., a transacting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention.
  • the nucleotide sequences encoding one or more of these factors are not necessarily in tandem with the nucleotide sequence encoding the polypeptide.
  • An expression vector typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or various activator genes.
  • the host cell of the present invention may be any cell capable of being genetically manipulated to express a polypeptide. Examples of host cells are given above.
  • transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the polypeptide When the modified polypeptide or the parent polypeptide of the present invention is expressed in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or it may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In t he I atter case, t he p olypeptide m ay b e recovered from t he p eriplasmic s pace b y disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.
  • ay b e recovered from t he p eriplasmic s pace b y disrupting the cells, e
  • the polypeptide When the modified or parent polypeptide is expressed in gram-positive bacteria such as Bacillus or Streptomyces strains, the polypeptide may be retained in the cytoplasm, or it may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the polypeptide may be recovered from the medium as described below. If the host cell is an insect cell transfection and production of recombinant polypeptides may be performed as described in US 4,745,051; US 4, 775, 624; US 4,879,236; US 5,155,037; US 5,162,222 or EP 397,485. Mammalian cells may for example be transfected by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546).
  • the above mentioned host cells transformed or transfected with a vector comprising a nucleotide sequence encoding a modified or parent polypeptide of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
  • the medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references for bacteria and yeast;
  • insect cells culture conditions may be as described in WO 89/01029 or WO 89/01028.
  • the modified or parent polypeptide of the present invention may be recovered directly from the medium. If it is not secreted, it may be recovered from cell lysates.
  • the modified and/or parent polypeptide of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulfate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chroma-tography, or the like, dependent on the polypeptide in question.
  • a salt e.g. ammonium sulfate
  • the modified and/or parent polypeptide of the invention may be detected using methods known in the art which are specific for the polypeptide. Examples of detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures or assays for determining different kinds of activity are known in the art.
  • the modified and/or parent polypeptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulphate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • SDS-polyacrylamide Gel electrophoresis may be used as qualitative analysis for the purity of a polypeptide using standard methods.
  • heterologous host cell When an expression vector comprising a nucleotide sequence encoding a modified or parent polypeptide of the present invention is transformed/transfected into a heterologous host cell it is possible to enable heterologous recombinant production of polypeptide.
  • An advantage of using a heterologous host cell is that it is possible to make a highly purified polypeptide composition, characterized in being free from homologous impurities, which are often present when a polypeptide is expressed in a homologous host cell.
  • homologous impurities mean any impurity (e.g. other polypeptides than the polypeptide of the invention) which originates from the homologous cell where the polypeptide of the invention is originally obtained from.
  • the method of the present invention results in increased expression of the modified polypeptide compared to expression of the parent polypeptide when both polypeptides are expressed under the same conditions.
  • increased expression is in the context of the present invention to be understood as the amount of modified polypeptide expressed by a given number of host cells and within a given period of time is higher than the amount of parent polypeptide expressed by the same number of host cells and within the same period of time and where expression of the modified and the parent polypeptide is performed under the same conditions.
  • shortening conditions refer to that the nucleotide sequences encoding the modified and the parent polypeptide are introduced by the same means into the same host cell, the host cell is cultured under the same conditions suitable for transcription and translation of said nucleotide sequences and the amount of polypeptides are measured by the same means.
  • the amount of polypeptide may be given in moles, or by weight, e.g. in grams, or by mass or by polypeptide activity, e.g. if the parent polypeptide is an enzyme it may be given as catalytic activity or if the parent polypeptide is an antibody or antigen or a receptor it may be given as binding affinity.
  • the parent and/or modified polypeptide may be purified as described above by e.g. conventional chromatographic methods or salting out effect.
  • SDS-Polyacrylamide Gel electrophoresis SDS-PAGE
  • SDS-PAGE SDS-Polyacrylamide Gel electrophoresis
  • the amount of a polypeptide may be quantified by amino acid analysis, as e.g. described in "Current Protocols in Protein science", Volume 2, edited by Coligan JE, Dunn BM, Ploegh HL, Spiecher D and Wingfield PT.
  • the amount of a purified polypeptide may also be determined by amino terminal analysis, i.e. sequencing the polypeptide, and mass spectral analysis and/or amino acid analysis.
  • Amino terminal analysis may e.g. be performed as described by Crabb JW et al. in "Current protocols in Protein Science” (1997) 11.9.11-11.9.42 (eds. Coligan JE et al.).
  • Several colorimetric methods are also known for quantification of the amount of a polypeptide, e.g. the Lowry method, as described in in Lowry ,O.H. et al. (1951) Protein measurements with the Folin phenol reagent. J.Biol.Chem 193,265-275.
  • Coomassie dye also can be used to estimate protein concentration as described in Bradford M (1976), A rapid and sensitive method for quantitation of micrograms quantities of protein utilizing the principle of protein dye binding, Anal. Biochim, 72, pp. 248-254
  • Fluorescence assays can be used to quantify the amount of a polypeptide using e.g. O- Phthaldehyde and Fmoc-chloride (9-Fluoroenyl methyl chloroformate) as described in Godel H.,Seitz P.and Verhoef M (1992), Automated amino acid analysis using combined OPA and Fmoc-CI precolumn derivatization, LC.GC International 5, pp. 44-49. If the parent polypeptide is an enzyme the amount of expressed modified and parent polypeptide may be measured by measuring the amount of enzymatic activity. Assays for measuring an enzymatic activity are as described above well known in the art.
  • the amount of a polypeptide may also be measured in e.g. an Immune precipitation assay using e.g. polyclonal antibodies against the polypeptide, such as single radial immuno diffusion assay, or Rocket immuno electrophoresis assay. Examples of these assays are described in Handbook of Immunoprecipitation-in gel techniques edited by Niels H. Axelsen Blackwell Scientific publications, Scandinavian Journal of Immunology Supplement, No. 10, Volume 17, 1983.
  • the amount of polypeptide may be measured by measuring the amount of lipase activity, which may be measured as described in " Methods for lipase detection and assay: a critical review", Beisson F et al., European Journal of Lipid Science and Technology, Vol. 102 (2) pp. 133-153 (2000) FEB.
  • the parent polypeptide is Phospholipase A2
  • the amount of polypeptide may be measured by measuring the enzymatic activity by monomolecular film technique as described by Ransac S et al., Eur.J.Biochem.204, 793-797 (1992). Another method involves measuring the release of fatty acids from lechitin by e.g. HPLC.
  • the p resent i nvention relates to a method for preparing a n ucleotide sequence encoding a modified polypeptide comprising: a) providing a nucleotide sequence encoding a parent polypeptide wherein said parent polypeptide comprises at least two Cys residues forming a disulfide bond; b) modifying at least one codon of the nucleotide sequence, wherein said codon specifies a Cys residue involved in a disulfide bond, so that the modified codon does not specify a Cys residue; and wherein said modification results in increased expression of the modified polypeptide as compared to expression of the parent polypeptide.
  • the method may further comprise any of the following steps: c) expressing the modified nucleotide sequence in a host cell d) isolating the modified polypeptide e) measuring the amount of modified polypeptide expressed by a host cell
  • Step c) expression of the modified nucleotide sequence in a host cell may comprise the steps of: i) introducing the modified nucleotide sequence into a host cell ii) culturing the host cell obtained in step i) under conditions suitable for expressing the modified polypeptide.
  • Step c) may typically take place after step b), step d) may typically take place after step c) and step e) may typically take place after step d).
  • the steps may be in another order, e.g. the method may further comprise step c) and e) but not step d).
  • the present invention also relates to a method for screening a library of modified nucleotide sequences for a nucleotide sequence having increased expression compared to expression of a parent nucleotide sequence.
  • library is intended to be understood as a collection of nucleotide sequences which differ at one or more nucleotide positions.
  • the present invention also relates to a method for screening a library of modified nucleotide sequences, comprising: a) providing a nucleotide sequence encoding a parent polypeptide wherein the parent polypeptide comprises at least two Cys residues forming a disulfide bond; b) generating a library of modified nucleotide sequences by modifying at least one codon of the nucleotide sequence provided in step a), wherein said codon specifies a Cys residue i nvolved i n a d isulfide b ond, s o that the modified codon does n ot specify a Cys residue; c) selecting a modified nucleotide sequence, wherein expression of said modified nucleotide sequence is increased as compare to expression of a parent nucleotide sequence.
  • the screening method may further comprise any of the following steps: ba) expressing the library of modified nucleotide sequences in a host cell; bb) isolating the modified polypeptide; be) measuring the amount of modified polypeptide expressed by a host cell.
  • Step ba) expression of the modified nucleotide sequence in a host cell may comprise the steps of: i) introducing the library of modified nucleotide sequences into a host cell ii) culturing the host cell obtained in step i) under conditions suitable for expressing the library of modified polypeptides.
  • Steps ba), bb) and be) may typically take place after step b) and before step c). However, it may also be possible for step bb) and/ or step be) to take place after step c).
  • the present invention also relates to a method for preparing a modified polypeptide, comprising: a) cultivating a host cell under conditions suitable for expression of a modified polypeptide, wherein the host cell comprises a nucleotide sequence which has been modified by modifying at least one codon specifying a Cys residue involved in a disulfide bond in a parent polypeptide so that said codon does not specify a Cys residue, and wherein said modification results in increased expression of the modified polypeptide as compared to expression of the parent polypeptide; b) recovering the modified polypeptide from the cultivation medium.
  • Lecithin-plates, pH 5.0 were prepared by: 10 g agar were melted in 0.1 M tri-Natriumcitrat-dihydrate buffer pH 5.0 (0.1 M is the finale concentration), 6 g lechitin (L-a-phosphatidylcholine 95%) and 2 ml 2% crystal violet were added to the solution and finally water was added to a finale volume of 1 Litre.
  • the solution was homogenized with Ultra-Turrax and then poured onto microtiter plate lids. Holes were punched in the lechitin-agar and 5 micro litre of media in which transformants have grown were added per hole. The plates are incubated at 37 degrees Celsius overnight. Transformants expressing phosphorlipase activity were identified as blue zones around the holes.
  • variants were obtained by site-directed mutagenesis of the corresponding nucleic acid sequences as described in for example Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbour, NY).
  • Apergillus oryzae was used as host cell
  • Phospholipase A2 from pig (Pig PLA2) (EMBL: SSPLA2R) comprising a S21R mutation (Pig PLA2; S21R) to ensure proper processing by a kex2-like protease found in Aspergillus oryzae.
  • Native Pig PLA2 and Pig PLA2; S21R comprise 7 disulfide bonds; C11-C77, C27-C123, C29- C45, C44-C105, C51-C98, C61-C91 and C84-C96 where the numbers are counted with the Methionine of the translation product as the first amino acid.
  • the Pig PLA2; S21R sequence is shown in SEQ ID NO. 1.
  • the nucleic acid sequence encoding Pig PLA2; S21R was modified by site-directed mutagenesis to create variants with one or more disulfide bonds disrupted.
  • the Pig PLA2 activity measured in the culture medium of A. oryzae transfected with the different Pig PLA2 variants is given in table 1 below.
  • the activity is evaluated subjectively by the presence (+) or absence (-) of a blue zone around the hole in the lechitin plate.
  • the presence of a blue zone indicates that the host cells express a phospholipase activity while the absence of a blue zone indicates that they do not express a phospholipase activity.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode de production d'un polypeptide modifié dans une cellule hôte consistant à rompre une ou plusieurs liaisons disulfure dans un polypeptide parent, ladite méthode entraînant une expression accrue du polypeptide modifié comparé au polypeptide parent.
PCT/DK2004/000597 2003-09-11 2004-09-10 Expression accrue d'un polypeptide modifie Ceased WO2005024012A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200301308 2003-09-11
DKPA200301308 2003-09-11

Publications (1)

Publication Number Publication Date
WO2005024012A1 true WO2005024012A1 (fr) 2005-03-17

Family

ID=34259080

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2004/000597 Ceased WO2005024012A1 (fr) 2003-09-11 2004-09-10 Expression accrue d'un polypeptide modifie

Country Status (1)

Country Link
WO (1) WO2005024012A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11162080B2 (en) 2007-03-30 2021-11-02 The Research Foundation For The State University Of New York Attenuated viruses useful for vaccines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036092A2 (fr) * 1998-12-17 2000-06-22 Biogen, Inc. Methode permettant d"obtenir un haut niveau d"expression de proteines chimeres immunoglobulines actives receptrices de la lymphotoxine-beta, et purification desdites proteines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036092A2 (fr) * 1998-12-17 2000-06-22 Biogen, Inc. Methode permettant d"obtenir un haut niveau d"expression de proteines chimeres immunoglobulines actives receptrices de la lymphotoxine-beta, et purification desdites proteines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BEYER B M ET AL: "Site-directed mutagenesis of a disulfide bridge in cathepsin D: expression, activation, purification, and characterization.", ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY. 1995, vol. 362, 1995, pages 285 - 287, XP008038855, ISSN: 0065-2598 *
WITTRUP K DANE: "Disulfide bond formation and eukaryotic secretory productivity", CURRENT OPINION IN BIOTECHNOLOGY, vol. 6, no. 2, 1995, pages 203 - 208, XP002305762, ISSN: 0958-1669 *
ZHOU S ET AL: "Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability", JOURNAL OF VIROLOGY, NEW YORK, US, US, vol. 66, no. 9, September 1992 (1992-09-01), pages 5393 - 5398, XP002961917, ISSN: 0022-538X *
ZHU HONGXIN ET AL: "Phospholipase A-2 engineering. The roles of disulfide bonds in structure, conformational stability, and catalytic function", BIOCHEMISTRY, vol. 34, no. 46, 1995, pages 15307 - 15314, XP002305763, ISSN: 0006-2960 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11162080B2 (en) 2007-03-30 2021-11-02 The Research Foundation For The State University Of New York Attenuated viruses useful for vaccines

Similar Documents

Publication Publication Date Title
CN108138362B (zh) 模块化多肽文库以及其制备和使用方法
US10202593B2 (en) Evolved sortases and uses thereof
US20170226495A1 (en) Sortase molecules and uses thereof
US6664092B1 (en) Polypeptides having dipeptidyl aminopeptidase activity and nucleic acids encoding same
US8569029B2 (en) DNase expression in recombinant host cells
DK3182830T3 (en) Use of GH5 xylanase and GH8, GH10 or GH11 xylanase in dough, dough and baking composition comprising these xylanases
AU2001244091B2 (en) Signal sequence trapping
JP2007535913A (ja) ヘテロカリオン菌類又は菌類宿主細胞におけるモノクローナル抗体の生成
CN110191641B (zh) 内酯酶及其使用方法
EP1230348A1 (fr) Criblages a haut rendement bases sur des plaques de microtitration
WO2005024012A1 (fr) Expression accrue d'un polypeptide modifie
US20220002690A1 (en) Polypeptides having lipase activity and use thereof for wheat separation
CN101578367B (zh) 良好表达的合成基因的选择
US20020019009A1 (en) High throughput screening (HTS) assays
CA2425380A1 (fr) Plantes transgeniques
AU779195B2 (en) High throughput screening (HTS) assays for protein variants with reduced antibody binding capacity
CA2531494A1 (fr) Procede de criblage permettant d'ameliorer l'activite specifique d'enzymes
WO2003046195A1 (fr) Procede pour produire une bibliotheque de variants specifique au site
US20030041354A1 (en) Transgenic plants
WO2001032834A1 (fr) Procede de criblage de populations de cellules
HK40067078A (en) Modular polypeptide libraries and methods of making and using same
HK1256697B (en) Modular polypeptide libraries and methods of making and using same
ZA200206216B (en) Signal sequence trapping.

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase