CN105377882A - Host cells and methods of use - Google Patents

Abstract

The present invention relates to genetically modified host cells, in particular yeast cells, comprising at least one isolated polynucleotide encoding a Killer Expression protease (Kex2p) or a fragment and/or variant thereof which has at least one Kex2p functional activity and at least one isolated polynucleotide encoding a Protein Disulfide-Isomerase (Pdil) or a fragment and/or variant thereof which has at least one Pdi functional activity. Also provided herein are genetically modified host cells comprising at least one isolated polynucleotide encoding a Killer Expression protease (Kex2p) or a fragment and/or variant thereof which has at least one Kex2p functional activity, at least one isolated polynucleotide encoding a Protein Disulfide-Isomerase (Pdil) or a fragment and/or variant thereof which has at least one Pdil functional activity and at least one isolated polynucleotide encoding a Endoplasmic Reticulum Oxidoreductin (Erol) or a fragment and/or variant thereof which has at least one Erol functional activity.

Description

Host cell and using method

Invention field

The invention belongs to biochemical engineering field.More specifically, the present invention relates to genetically modified host cell and the method for producing polypeptide in them.

Background of invention

Can in multiple host cell express therapeutic peptide and protein, comprise bacterial cell, Bacillus coli cells, fungi or yeast cell, microbial cell, insect cell and mammalian cell.Fungal host such as methylotrophic yeast pichia pastoris phaff (Pichiapastoris) has the unique advantage of being used for the treatment of property protein expression: such as, it does not secrete a large amount of endogenous proteins, it has stronger inducible promoters, it can determine to grow in Components Chemical substratum, and it can produce the recombinant protein (Cregg etc., Mol.Biotech.16:23-52 (2000)) of high titre.Yeast and filamentous fungus are used successfully to and have produced in born of the same parents and recombinant protein (Cereghino, J.L. and the J.M.Cregg2000FEMSMicrobiologyReviews24 (1): 4566 of secretion; Harkki, A., wait 1989Bio-Technology7 (6): 596; Berka, R.M., wait 1992Abstr.PapersAmer.Chem.Soc.203:121-BIOT; Svetina, M., wait 2000J.Biotechnol.76 (23): 245-251.Yeast saccharomyces cerevisiae is a kind of outstanding host cell for expressing recombinant human serum white protein (HSA).But, express the technology barrier comprising the low titre being faced with recombinant protein with the other treatment polypeptide of the polypeptide of HAS genetic fusion.

So, the host cell, particularly Wine brewing yeast strain that can produce heterologous peptides, polypeptide and/or protein with high titre recombinant protein is needed.

Summary of the invention

In one aspect of the invention, genetically modified host cell is provided, it comprises coding and kills and wounds expressing protein enzyme (KillerExpressionprotease, or the polynucleotide that are separated of at least one with its fragment of at least one Kex2p functionally active and/or variant Kex2p), and the polynucleotide that proteins encoded disulphide isomerase (ProteinDisulfide-Isomerase, Pdi1) or at least one with its fragment of at least one Pdi functionally active and/or variant are separated.Additionally provide genetically modified host cell herein, the polynucleotide that its at least one comprising its fragment and/or variant that coding kills and wounds expressing protein enzyme (Kex2p) or has at least one Kex2p functionally active is separated, the polynucleotide that proteins encoded disulphide isomerase (Pdi1) or at least one with its fragment of at least one Pdi functionally active and/or variant are separated, and the polynucleotide that coding endoplasmic reticulum redox element (Oxidoreductin) (Ero1) or at least one with its fragment of at least one Ero1 functionally active and/or variant are separated.

In yet another aspect, the invention provides genetically modified host cell, when cultivating described genetically modified host cell in culture, the at least one gene product of polynucleotide that it is expressed or described in process LAN, at least one is separated, described polynucleotide encoding is selected from following protein or has its fragment of at least one functionally active and/or the variant of described protein: Kex2p, Pdi1 and Ero1.Another aspect of the present invention provides genetically modified host cell, when cultivating described genetically modified host cell in culture, it is selected from least two kinds of protein of Kex2p, Pdi1 and Ero1 or its fragment with at least one functionally active of described at least two kinds of protein and/or variant compared to wild-type host cells process LAN, wherein said wild-type host cells belongs to same species and cultivates under same culture conditions, but process LAN is not selected from least two kinds of gene products of Kex2p, Pdi1 and Ero1.Host cell can be protokaryon or eucaryon.The example of host cell can include but not limited to: HeLa, CHO, COS, HEK293, THPI, yeast and insect cell.In particular embodiments, mammalian cell is hamster, people or mouse cell.In a specific embodiment, cell is Chinese hamster ovary celI system, HEK293 clone or bhk cell system.

The method producing recombinant polypeptide is also provided herein, comprises and cultivate host cell of the present invention.In yet another aspect, the invention provides the recombinant polypeptide prepared by method of the present invention.Go back the pharmaceutical composition of providing package containing the recombinant polypeptide prepared by method of the present invention herein.In another aspect of the present invention, the method for the treatment of the patient having this to need is provided, comprises the pharmaceutical composition of the present invention of administering therapeutic significant quantity.

Accompanying drawing is sketched

Fig. 1: the preparation master cell bank (PreliminaryMasterCellBank) creating the bacterial strain producing albiglutide (albiglutide).The step of producing host from KEX2-KanMX expression cassette PCR primer to BXP10KEX2PDIERO1 is that sequential integration expression box is with building process IV (ProcessIV) host strain BXP10_KEX2_PDI_ERO1.After conversion pCID3610 plasmid, select final production clone and for the preparation of preparation master cell bank (PCB).

Fig. 2: the Southern engram analysis of PDI1 and KEX2 in host strain.Endogenous KEX2 and PDI1 gene are arranged in chromosome x IV and III with single copy (wild-type) respectively.The target site of integration is shown under wild-type in chromosome x II.The detection probes of each gene is shown as Filled Rectangle.

Fig. 3: from the western blot analysis of Pdi1 and Kex2p of host strain.Sample in road, 1-5 road: 5 clones of BXP10-KEX2-PDI1 bacterial strain; PDI: the BXP10 of process LAN Pdi1; KEX2: the BXP10 of process LAN Kex2p; And BXP10: host strain in contrast.Load the protein of equivalent.

Fig. 4: from the SDS-PAGE of 12 parts of supernatant samples of oscillating flat plate test.Road in gel; The prestained protein ladder (Invitrogen) of L:SeeBlue2; The reference standard of RS:pCID3610 albumen; 1-12: 12 subclones of expressing pCID3610.

Fig. 5: the titre (A) of pCID3610 albumen produced in DasGip fermentation runs and the analysis of quality (B).The supernatant liquor titre productive rate (yield) of pCID3610 albumen and 6-AA level (%) are compared with BXP10-KEX2-PDI1 in contrast, the latter is the BXP10 of process LAN Kex2p and Pdi1.

Fig. 6: from the growth curve of research cell bank bottle (ResearchCellBankVial) Hemapoiesis.

Fig. 7: from the growth curve of preparation master cell bank (Pre-MasterCellBank) Hemapoiesis.

Detailed Description Of The Invention

" host cell " refers to import (such as transform, infect or transfection) separative polynucleotide sequence maybe can import (such as transform, infect or transfection) cell with the polynucleotide sequence be separated as used in this article.Host cell of the present invention can include but not limited to, bacterial cell, fungal cell, yeast cell, microbial cell, insect cell and mammalian cell.The host cell with yeast and/or filamentous fungus origin of the present invention can include but not limited to following section, belong to and plant: pichia pastoris phaff, Pichiafinlandica, Pichiatrehalophila, Pichiakoclamae, Pichiamembranaefaciens, pichia methanolica (Pichiamethanolica), Pichiaminuta (Ogataeaminuta, Pichialindneri), Pichiaopuntiae, Pichiathermotolerans, Pichisalictaria, Pichiaguercum, Pichiapijperi, Pichiastiptis, Pichia bacterial classification (Pichiasp.), Saccharomycescastelii, yeast saccharomyces cerevisiae (Saccharomycescerevisiae), kluyveromyces (Saccharomyceskluyveri), Saccharomyces sp (Saccharomycessp.), schizosaccharomyces pombe (Schizosaccharomycespombe), Japan fission yeast (Schizosaccharomycesjaponicus), eight spore fission yeasts (Schizosaccharomycesoctosporus), Schizosaccharomycescryophilus, Schizosaccharomyces bacterial classification (Schizosaccharomycessp.), multiple-shaped nuohan inferior yeast (Hansenulapolymorpha), genus kluyveromyces bacterial classification (Kluyveromycessp.), Kluyveromyces lactis (Kluyveromyceslactis), Candida albicans (Candidaalbicans), mycocandida bacterial classification (Candidasp.), Aspergillus fumigatus (Aspergillusfumigatus), Aspergillus nidulans (Aspergillusnidulans), aspergillus niger (Aspergillusniger), aspergillus oryzae (Aspergillusoryzae), Trichodermareesei (Trichodermareesei), Chrysosporiumlucknowense, sickle mycete kind (Fusariumsp.), Fusariumgramineum, empiecement sickle spore (Fusariumvenenatum), Physcomitrellapatens, Yarrowia lipolytica (Yarrowialipolytica), Arxulaadeninivorans, prosperous yeast (Schwanniomycesoccidentalis) is permitted in west, with Neurospora crassa (Neurosporacrassa).

As known in the art, " conversion " is via the importing of outside DNA or RNA to the genome of organism or episomal directed modification, or any other of outside DNA or RNA is stable imports.

As known in the art, " transfection " is that outside DNA or RNA (including but not limited to recombinant DNA or RNA) is imported microorganism.

As known in the art, " identity " is the relation between two or more peptide sequences or two or more polynucleotide sequences (depending on the circumstances), as determined by sequence as described in relatively.In the art, " identity " also means the serial correlation degree between polypeptide or polynucleotide sequence (depending on the circumstances), as determined in the coupling between the string by this kind of sequence.Easily calculate " identity " by currently known methods, include but not limited to those (ComputationalMolecularBiology, Lesk, A.M. volume, OxfordUniversityPress, NewYork, 1988 described below; Biocomputing:InformaticsandGenomeProjects, Smith, D.W. compile, AcademicPress, NewYork, 1993; ComputerAnalysisofSequenceData, PartI, Griffin, A.M. and Griffin, H.G. compiles, HumanaPress, NewJersey, 1994; SequenceAnalysisinMolecularBiology, vonHeinje, G., AcademicPress, 1987; And SequenceAnalysisPrimer, Gribskov, M. and Devereux, J. compiles, MStocktonPress, NewYork, 1991; And Carillo, H. and Lipman, D., SIAMJ.AppliedMath., 48:1073 (1988).Measure the maximum match of method design for providing between cycle tests of identity.And the method measuring identity is compiled in the computer program that openly can obtain.The computer program means measuring the identity between two sequences includes but not limited to, GCG routine package (Devereux, J., Deng, 387 (1984)), BLASTP, BLASTN and FASTA (Altschul NucleicAcidsResearch12 (1):, S.F. etc., J.Molec.Biol.215:403-410 (1990).BLASTX program openly can obtain from NCBI and other sources (BLASTManual, Altschul, S., etc., NCBINLMNIHBethesda, MD20894; Altschul, S., etc., J.Mol.Biol.215:403-410 (1990).Famous SmithWaterman algorithm can also be used to measure identity.

The parameter compared for peptide sequence comprises following: algorithm: Needleman and Wunsch, J.MolBiol.48:443-453 (1970) comparator matrix: from the BLOSSUM62 of Hentikoff and Hentikoff, Proc.Natl.Acad.Sci.USA.89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

With these parameters can procedure publication can obtain as " gap " program from GeneticsComputerGroup, MadisonWI.Aforementioned parameters is the default parameter (and no penalty for end gaps) compared for peptide.

The parameter compared for polynucleotide comprises following: algorithm: Needleman and Wunsch, J.MolBiol.48:443-453 (1970)

Comparator matrix: coupling=+ 10, erroneous matching=0

Gap Penalty: 50

Gap Length Penalty: 3

Obtain: from " gap " program of GeneticsComputerGroup, MadisonWI.These are the default parameters compared for nucleic acid.

The implication of " identity " of polynucleotide and polypeptide (depending on the circumstances) is provided below in (1) and (2).

(1) polynucleotide embodiment also comprises comprising and has at least 50 with canonical sequence (such as SEQIDNO:3), 60, 70, 80, 85, 90, 95, 97 or 100% polynucleotide of separation of polynucleotide sequence of identity, wherein said polynucleotide sequence can be identical with the canonical sequence of SEQIDNO:3, or the Nucleotide that can comprise nearly specific integer number compared with canonical sequence changes, wherein said change is selected from lower group: at least one nucleotide deletion, replace (comprising conversion and transversion) or insert, wherein said change can occur in reference to any place between 5' or the 3' terminal position of nucleotide sequence or those terminal positions, individually be dispersed in the Nucleotide in canonical sequence or be dispersed in one or more continuous group in canonical sequence, and wherein said Nucleotide changes number by the total nucleotide number in SEQIDNO:3 being multiplied by the integer division of definition percentage identities with 100, then deduct this product to determine from the total nucleotide number described SEQIDNO:3, or:

nn≤xn-(xn·y),

Wherein n _nthe number that Nucleotide changes, x _nbe the total nucleotide number in SEQIDNO:3, y is 0.95 for 95%, is 0.97 for 97%, is 1.00 for 100%, is the symbol of multiplication operator, and wherein x _nwith any non-integer product of y from x _nrounded downwards to immediate integer before deducting it.The change of the polynucleotide sequence of coded polypeptide can create nonsense, missense or phase shift mutation in this encoding sequence, changes afterwards by the polypeptide of polynucleotide encoding thus in this kind of change.

(2) polypeptide embodiment also comprises isolated polypeptide, it comprises and has at least 50 with polypeptide canonical sequence such as SEQIDNO:1, 60, 70, 80, 85, 90, 95, 97 or 100% polypeptide of identity, wherein said peptide sequence can be identical with canonical sequence, or the amino acid change of nearly specific integer number can be comprised compared with canonical sequence, wherein said change is selected from lower group: at least one aminoacid deletion, replace (comprising conservative property and non-conservation replacement) or insert, wherein said change can occur in reference to any place between the amino of peptide sequence or carboxy terminal positions or those terminal positions, individually be dispersed in the amino acid in canonical sequence or be dispersed in one or more continuous group in canonical sequence, and wherein said amino acid change number is by being multiplied by the integer division of definition percentage identities with 100 by amino acid sum, then deduct this product to determine from described amino acid sum, or:

na≤xa-(xa·y),

Wherein n _athe number of amino acid change, x _abe the amino acid sum in sequence, y is 0.95 for 95%, is 0.97 for 97%, is 1.00 for 100%, is the symbol of multiplication operator, and wherein x _awith any non-integer product of y from x _arounded downwards to immediate integer before deducting it.

" separation " means, and " by artificial " changes from its native state, if namely it is present in occurring in nature, it is changed from its initial environment or takes out, or both.Such as, naturally be present in polynucleotide in live organism or polypeptide is not " separation ", the same polynucleotide be separated from the coexisting materials of its native state or polypeptide are only " separation ", include but not limited to, when this kind of polynucleotide or polypeptide be imported into get back in cell time.

" separation " or " substantially pure " nucleic acid or polynucleotide (polymkeric substance of such as RNA, DNA or mixing) are and the natural nucleic acid that is substantially separated in its native host cell, with other cellular components (such as rrna, polysaccharase and genome sequence) of its associated in nature with native polynucleotide or polynucleotide.Following nucleic acid or polynucleotide contained in this term, its (1) takes out from its naturally occurring environment, (2) all or part of associating of the polynucleotide of " polynucleotide be separated " with is not wherein naturally found, (3) be operably connected at its unconnected polynucleotide of occurring in nature, or (4) do not exist at occurring in nature.Term " separation " or " substantially pure " also can be used for the polynucleotide analogue referring to the DNA isolate of restructuring or clone, the polynucleotide analogue of chemosynthesis or synthesized by Heterologous System biology.

But " separation " not necessarily requires that the nucleic acid of description like this or polynucleotide itself physically take out from its natural surroundings.Such as, if heterologous sequence is placed near endogenous nucleotide sequence, the expression of this endogenous nucleotide sequence is changed (such as increase, reduce or eliminate), and this endogenous nucleotide sequence so in organism genome is regarded as " separation " in this article.In this context, heterologous sequence is the not natural sequence being close in endogenous nucleotide sequence, and no matter this heterologous sequence itself is endogenous (being derived from identical host cell or its offspring) or external source (being derived from different hosts cell or its offspring).For example, promoter sequence can replace the natural promoter (such as passing through homologous recombination) of gene in host cell gene group, thus makes this gene have the expression pattern of change.This gene now becomes " separation ", because its at least some sequence of itself and natural side joint is separated.

If nucleic acid contains the not natural any modification occurred in genome in corresponding nucleic, then this nucleic acid is also regarded as " separation ".Such as, if interior source coding sequence contain manually (such as intervened by people) introduce insertion, disappearance or point mutation, then this sequence is regarded as " separation "." nucleic acid of separation " is also included in the nucleic acid construct that heterologous site is incorporated into the nucleic acid in host cell chromosome and exists as episome.And " nucleic acid of separation " can not have other cell materials substantially, does not maybe substantially have substratum when being produced by recombinant technology, or substantially there is no precursor or other chemicals when chemosynthesis.

As used in this article, " nucleotide sequence of encoding function gene product " refers to the arbitrary portion of genes encoding part.The functional gene product of nucleic acid sequence encoding can be a part for enzyme, and its at least one that can realize holoenzyme or complete enzyme is active.

As used in this article, " express the nucleic acid that at least one gene product is required " and refer to encoding gene arbitrary portion and/or be operably connected to the nucleic acid of encodes gene product but not necessarily comprise the nucleotide sequence of encoding sequence.Such as, the nucleotide sequence of expressing at least one gene product required includes but not limited to, enhanser, promotor, regulating and controlling sequence, beginning codon, terminator codon, Polyadenylation sequences and/or encoding sequence.

As used in this article, " proteolysis " or " being responsible for the gene product of proteolysis in cell " refers to any peptides of the cutting that can cause at least one peptide, polypeptide and/or protein, polypeptide, protein and/or enzyme or its part.The gene product being responsible for proteolysis can be directly be responsible for cutting (i.e. peptase), or it can be responsible for indirectly as a part for peptase route of synthesis.The example being responsible for the gene product of proteolysis in cell includes but not limited to, the aspartyl protease of aspartyl protease, serine protease, secretion, the serine protease of secretion, yeast Methylotrophic proteolytic enzyme, DPPIV sample endopeptidase, Zinc metalloproteinase, Prb1 sample serine protease, Prb1 serine protease and CPY sample carboxypeptidase.Further, included within this definition is from emiocytosis, but still can retain the proteolytic enzyme of its some or all of proteolytic activity, as the serine protease of secretion.The proteolytic enzyme of secretion can be responsible in cell and/or the proteolysis of outside.

As used in this article, " glycosylation " or " be responsible for cell in glycosylated gene product " reference and in cell at least one sugar module to any peptides of the prolongation of the interpolation of polypeptide or at least one sugar chain, polypeptide, protein and/or enzyme or its part.Being responsible for glycosylated gene product in cell can be directly be responsible for the interpolation of sugar to polypeptide in cell, such as but not limited to mannose transferase.Residue from Dol-P-Man can be transferred to Serine on peptide, polypeptide and/or protein and/or threonine residues by mannose transferase, or can act on the mannose residue from GPD-Man is transferred to sugar, so extends sugar chain.Or, be responsible for part that glycosylated gene product can be glycosylation pathway differ and indirectly can be responsible in cell polysaccharide to the interpolation of polypeptide.The example being responsible for glycosylated gene product in cell includes but not limited to mannose transferase.

" polynucleotide " refer generally to any polybribonucleotide or polydeoxyribonucleotide, and it can be not modified RNA or DNA or modified RNA or DNA." polynucleotide " include but not limited to, strand and double-stranded DNA, as the DNA of strand and double stranded region or strand, double-strand and three sequence mixtures, strand and double-stranded RNA, with the RNA as strand and double stranded region mixture, comprise the heterozygote molecule of DNA and RNA, it for strand or can be more typically double-strand or three sequences or strand and double stranded region mixture.In addition, " polynucleotide " refer to three sequences comprising RNA or DNA or RNA and DNA as used in this article.Chain in this kind of region can from same molecular or from differing molecular.Region can comprise one or more whole molecules, but more generally only relates to certain region of some molecules.One of the molecule in triple helices district is often oligonucleotide.As used in this article, term " polynucleotide " also comprises above-described DNA or RNA comprising one or more modified bases.So, DNA or RNA with the adorned main chain for stability or other reasons is " polynucleotide ", as being intended in this article in this term.And DNA or RNA comprising uncommon base (as inosine) or modified base (as tritylated bases) (only for two examples) is polynucleotide, as used in this article in this term.To understand and carry out a variety of modification to DNA and RNA, it meets many useful objects well known by persons skilled in the art.This kind of form of modifying through chemistry, enzyme or metabolism of polynucleotide contained in term " polynucleotide " when it adopts in this article, and the chemical species of virus and cell (comprising such as simple and complex cell) distinctive DNA and RNA." polynucleotide " also contain the short polynucleotide being often called oligonucleotide.

" polypeptide " refers to comprise two or more amino acid whose any peptides or protein of being connected to each other by peptide bond or modified peptide bond." polypeptide " refers to often be called the short chain of peptide, oligopeptides and oligomer and be commonly referred to as the more long-chain of protein.Polypeptide can comprise the amino acid except 20 kinds of gene coding amino acids." polypeptide " comprise modified by natural process (as processing and other posttranslational modifications) those and modified by chemical modification technology those.This kind of basic reader that is modified at neutralizes and is documented in monograph and a large amount of Research Literature more specifically, and it well known to a person skilled in the art.The several sites being modified at given polypeptide that high-ranking military officer understands same type can identical or different degree exist.Further, given polypeptide can comprise many modified types.Modification can occur in the arbitrary place in polypeptide, comprises peptide main chain, amino acid side chain and amino or C-terminal.Modification comprises, such as acetylize, acidylate, ADP-ribosylation, amidation, the covalency attachment of flavine, the covalency attachment of protoheme module, the covalency attachment of Nucleotide or nucleotide derivative, the covalency attachment of lipid or lipid derivate, the covalency attachment of phosphatidylinositols, crosslinked, cyclisation, disulfide formation, demethylation, the formation of covalent cross-linking, the formation of halfcystine, the formation of Pyrrolidonecarboxylic acid, formylation, gamma-carboxylation, GPI anchor is formed, hydroxylation, iodate, methylate, myristoylation, oxidation, proteolysis is processed, phosphorylation, prenylation, racemization (racemization), glycosylation, lipid is attached, sulfation, the gamma-carboxylation of glutaminic acid residue, hydroxylation and ADP-ribosylation, selenizing (selenoylation), sulfation, what transfer RNA mediated adds amino acid to protein, such as arginyl (arginylation), with ubiquitination (ubiquitination).See, such as PROTEINS-STRUCTUREANDMOLECULARPROPERTIES, 2ndEd., T.E.Creighton, W.H.FreemanandCompany, NewYork (1993) and Wold, F., PosttranslationalProteinModifications:PerspectivesandPro spects, pgs.1-12inPOSTTRANSLATIONALCOVALENTMODIFICATIONOFPROTEIN S, B.C.Johnson compiles, AcademicPress, NewYork (1983); Seifter etc., Meth.Enzymol.182:626-646 (1990) and Rattan etc., ProteinSynthesis:PosttranslationalModificationsandAging, Ann.N.Y.Acad.Sci.663:48-62 (1992).When carrying out or do not carry out a point branching (branching), polypeptide can be branch or ring-type.Ring-type, branch or branched cyclic polypeptides can be derived from the rear natural process of translation, and prepare by complete synthetic method.

" variant " is different from reference polynucleotide or polypeptide difference when this term uses in this article but retains polynucleotide or the polypeptide of fundamental characteristics.The typical variant of polynucleotide is different on nucleotide sequence with reference to polynucleotide from another.Change in Variant nucleotide sequences can change or not change the aminoacid sequence by the polypeptide with reference to polynucleotide encoding.Nucleotide changes the aminoacid replacement that can cause in the polypeptide of being encoded by canonical sequence, interpolation, disappearance, fusion and brachymemma, as discussed below.The typical variant of polypeptide is different in aminoacid sequence with reference to polypeptide from another.Generally speaking, difference is limited, thus makes with reference to polypeptide extremely similar generally with the sequence of variant, and identical in many regions.Variant and one or more replacements of arbitrary combination, interpolation, disappearance can be with reference to polypeptide difference in aminoacid sequence.The amino-acid residue replaced or insert can the yes or no residue of being encoded by genetic code.The variant of polynucleotide or polypeptide can be naturally occurring (as allele variant), or it can be known not naturally occurring variant.The present invention also comprises the variant of every peptide species of the present invention, and it is with the difference of canonical sequence the polypeptide that conserved amino acid replaces, and wherein residue is replaced by another residue with similar characteristics.Typically this kind ofly be substituted by Ala, Val, Leu and Ile; In Ser and Thr; In acidic residues Asp and Glu; In Asn and Gln; With in alkaline residue Lys and Arg; Or in aromatic residue Phe and Tyr.Particularly, have that wherein several (5-10,1-5,1-3,1-2 or 1) amino acid is replaced with arbitrary combination, disappearance or the variant that adds.The variant that the non-natural of polynucleotide and polypeptide exists can be prepared by induced-mutation technique or direct synthesis.Variant also can include but not limited to, has polypeptide or its fragment of the chemically modified of its amino acid side groups of one or more.Chemically modified includes but not limited to, adds chemical module, creates new key, and removing chemical module.The modification at amino acid side groups place includes but not limited to, the acidylate of Methionin-epsilon-amino group, the N-alkylation of arginine, Histidine or Methionin, the alkylation of L-glutamic acid or aspartic carboxylic acid groups, and the desamidization of glutamine or l-asparagine.The modification of terminal amino group includes but not limited to, de--amino, N-low alkyl group (alkyl), N-be two-and low alkyl group and the modification of N-acyl group.The modification of terminal carboxyl groups includes but not limited to, acid amides, lower alkyl, dialkyl amide and lower alkyl esters are modified.In addition, by having the known protectiveness group of the protein chemistry man of ordinary skill to protect one or more side-chain radicals or end group.

As used in this article, " fragment " refer to have when mentioning polypeptide and using with complete natural there is polypeptide part but not the polypeptide of the identical aminoacid sequence of whole aminoacid sequence.As used in this article " fragment " mention polynucleotide or nucleotide sequence use out-of-date, the polynucleotide of the following aminoacid sequence that refers to encode, described aminoacid sequence and complete naturally there is the part of polypeptide but not the part of whole aminoacid sequence.Fragment can be " independently (free-standing) " or be included in larger polypeptide, and wherein this fragment forms a part or region as the single successive zone in single larger polypeptide.For example, the fragment of naturally occurring GLP-1 will comprise the amino acid 7 to 36 of naturally occurring amino acid/11 to 36.In addition, the fragment of polypeptide can also be the variant of naturally occurring partial sequence.Such as, the GLP-1 fragment comprising the amino acid 7-36 of naturally occurring GLP-1 can also be the variant in its partial sequence with aminoacid replacement.Again for example, " fragment " can refer to the nucleic acid (including but not limited to Kex2p, Pdi1 and Ero1) of any heterologous polypeptide described herein or coding said polypeptide, and wherein said fragment retains at least one functionally active of described wild type peptide or enzyme.

As used in this article, " to put together " or " puting together " refers to two molecules being bonded to each other.Such as, the first polypeptide can covalently or non-covalently in conjunction with the second polypeptide.First polypeptide be covalently bonded in by chemical linker or can genetic fusion in the second polypeptide, wherein the first and second polypeptide share total polypeptide backbone.The recombinant polypeptide of expressing in host cell of the present invention can comprise at least one therapeutical peptide puted together in human serum albumin.Other conjugates can include but not limited to, put together at least one therapeutical peptide at least one the Fc district in Transferrins,iron complexes, strand variable domain and/or antibody.Conjugate can comprise or not comprise joint.

As used in this article, " series connection orientation " refers to as same a part part two or more polypeptide adjacent one another are.They can covalency or non-same valency connection.The polypeptide of two or more series connection orientations can form the part of same polypeptide backbone.The polypeptide of series connection orientation can have directly or reversing orientation and/or can by other aminoacid sequences separately.

As used in this article, " albiglutide " refers to a kind of recombination fusion protein, it is by 2 copy compositions of 30 aminoacid sequences (GLP-1, fragment 7-36 (A8G)) of that merge with recombinant human serum continuous inheritance, modified human glucagon-like-peptide 1.The aminoacid sequence of albiglutide is shown as SEQIDNO:1 as follows.

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTSDVSSYLEGQAAKEFIAWLVKGR60

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAE120

NCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPE180

VDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLL240

PKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLT300

KVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMP360

ADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEK420

CCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVS480

TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTE540

SLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKA600

TKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL674

(SEQIDNO：1)

" recombinant expression system " guides, transfection or be transformed in host cell or host cell lysats to produce the expression system of the present invention of polynucleotide of the present invention and polypeptide or its part or polynucleotide.

As used in this article, " albumin fusion proteins " comprises at least one fragment of therapeutical peptide or at least one fragment of variant and human serum albumin or variant, and they combine with one another, preferably by gene fusion.

The polypeptide with GLP-1 activity can comprise at least one fragment and/or the variant of people GLP-1.Two of people GLP-1 are natural to be existed fragment and is shown in SEQIDNO:2.

Wherein: the Xaa being positioned at the 37th is that Gly is (hereinafter referred to " GLP-1 (7-37) ") ， Huo – NH ₂(hereinafter referred to " GLP-1 (7-36) ").GLP-1 fragment can be including but not limited to, comprises or form the GLP-1 molecule of the amino acid 7 to 36 (GLP-1 (7-36)) for people GLP-1.The variant of GLP-1 or its fragment can including but not limited to 1,2,3,4,5 in wild-type GLP-1 or in the naturally occurring GLP-1 fragment of SEQIDNO.:2 display an or more aminoacid replacement.Variant GLP-1 or GLP-1 fragment can include but not limited to, be similar to the replacement of the alanine residue of the L-Ala 8 of wild-type GLP-1, this kind of L-Ala is sported glycine (hereinafter referred to " A8G ") (see such as, U.S. Patent No. 5,545, suddenling change disclosed in 618, stating complete being incorporated to herein by carrying).

As used in this article, " KEX2 " refers to that coding is called " killing and wounding expressing protein enzyme " or the gene of the protein of " Kex2p " (being also called " kexp ") herein.Kex2p relates to the Ca-dependent serine protease that proteinogen (proprotein) is processed.This proteolytic enzyme is at the carboxyl terminal cutting polypeptide of recognition sequence Arg-Arg/X and Lys-Arg/X.Other Kex2p activity include but not limited to, hydrolytic enzyme activities, metal ion binding activities, serine-type endopeptidase activity, peptidase activity and serine-type peptidase activity.The assumed name of KEX comprises: Pcsk2, Pcsk4, kpc-1.Yeast saccharomyces cerevisiae endopeptidase (KEX2) gene has GenBankIDNo.855483 and coding NCBI protein sequence RefSeqNP:014161.1.This KEX2 gene is conservative in fruit bat, yeast saccharomyces cerevisiae, Kluyveromyces lactis (K.lactis), E.gossypii, schizosaccharomyces pombe (S.pombe), M.oryzae and Neurospora crassa (N.crassa).The variant of KEX2 can have at least 85%, 90%, 95% with from yeast saccharomyces cerevisiae (KEX2) gene, 96%, 97%, 98%, the polynucleotide of 99% sequence iden, or coding has and has at least 85%, 90%, 95% with the Kex2p from yeast saccharomyces cerevisiae, 96%, 97%, 98%, the protein of the aminoacid sequence of 99% sequence iden.The function fragment of Kex2p and/or variant, by retaining at least one function of Kex2p, include but not limited to, in the ability of the carboxyl terminal cutting polypeptide of recognition sequence Arg-Arg/X and Lys-Arg/X.Show below from the gene order (SEQIDNO:3) of the KEX2 of yeast saccharomyces cerevisiae and the corresponding aminoacid sequence (SEQIDNO:4) of Kex2p:

Source yeast saccharomyces cerevisiae

Source yeast saccharomyces cerevisiae

As used in this article, " PDI " or " PDI1 " refers to encode and is also called the gene of " pdi " or " Pdi1p " of " protein disulfide-isomerase ", this protein disulfide-isomerase is formation and the fracture (WilkinsonB of disulfide linkage in Eukaryotic endoplasmic reticulum when protein folding in catalytic proteins between cysteine residues, GilbertHF (June2004). " Proteindisulfideisomerase " .BiochimicaetBiophysicaActa1699 (1-2): 35 – 44 and GruberCW, CemazarM, HerasB, MartinJL, CraikDJ (August2006). " Proteindisulfideisomerase:thestructureofoxidativefolding " .TrendsinBiochemicalSciences31 (8): 455 – 64)) and molecular chaperone protein (Wang can be served as, CC and Tsou, CLFASEBJ.1993Dec, 7 (15): 1515-7) enzyme.Protein disulfide-isomerase is a kind of multifunctional protein be present in endoplasmic, is that the disulfide linkage that formation is secreted in property and cell surface proteins is necessary, unties (unscramble) non-native disulfide bond; Form mixture with the Mnl1p with Exo-Mannosidase activity, described Exo-Mannosidase is active is processed into Man7GlcNAc2 by the Man8GlcNAc2 oligosaccharides that unfolded protein combines, and this promotes the degraded in unfolded protein response.Pdi1 also has oxidoreductase activity.Pdi1p from yeast saccharomyces cerevisiae is encoded by GenBankIDNO.850314.The function fragment of pdi and/or variant can be have at least 85%, 90%, 95% with the aminoacid sequence of NCBIRefSeqNP_009887,96%, 97%, 98%, the polypeptide of 99% sequence iden, it has at least 85%, 90% by with GenBankIDNo.850314,95%, 96%, 97%, the polynucleotide encoding of 98%, 99% sequence iden, this polypeptide will retain at least one function of Pdi, include but not limited to, isomerase activity.Pdi1 functionally active includes but not limited to, the formation of catalyses disulfide bond and/or fracture, helps the correct folding of misfolded protein matter.The assumed name of PDI comprises: PDI1, PDIA2, Pdia3, P4HB, PADI1, Padi2, EUG1, NCU09223, SOAC1F5.02 and AGOS_AFR718W.PDI1 gene is conservative in people, rhesus monkey, dog, ox, mouse, rat, chicken, zebra fish, fruit bat, mosquito, Caenorhabditis elegans (C.elegans), yeast saccharomyces cerevisiae, Kluyveromyces lactis (K.lactis), E.gossypii, schizosaccharomyces pombe (S.pombe), M.oryzae, Neurospora crassa (N.crassa), Arabidopis thaliana (A.thaliana) and rice.(PDI1) function can be played.Show below from the gene order (SEQIDNO:5) of the PDI of yeast saccharomyces cerevisiae and the corresponding aminoacid sequence (SEQIDNO:6) of Pdi1:

Source yeast saccharomyces cerevisiae

Source yeast saccharomyces cerevisiae

MKFSAGAVLSWSSLLLASSVFAQQEAVAPEDSAVVKLATDSFNEYIQSHDLVLAEFFAPWCGHCKNMAPEYVKAAETLVEKNITLAQIDCTENQDLCMEHNIPGFPSLKIFKNSDVNNSIDYEGPRTAEAIVQFMIKQSQPAVAVVADLPAYLANETFVTPVIVQSGKIDADFNATFYSMANKHFNDYDFVSAENADDDFKLSIYLPSAMDEPVVYNGKKADIADADVFEKWLQVEALPYFGEIDGSVFAQYVESGLPLGYLFYNDEEELEEYKPLFTELAKKNRGLMNFVSIDARKFGRHAGNLNMKEQFPLFAIHDMTEDLKYGLPQLSEEAFDELSDKIVLESKAIESLVKDFLKGDASPIVKSQEIFENQDSSVFQLVGKNHDEIVNDPKKDVLVLYYAPWCGHCKRLAPTYQELADTYANATSDVLIAKLDHTENDVRGVVIEGYPTIVLYPGGKKSESVVYQGSRSLDSLFDFIKENGHFDVDGKALYEEAQEKAAEEADADAELADEEDAIHDEL(SEQIDNO：6)

PDI is the permanent albumen in endocytoplasmic reticulum chamber.A series of evidences on enzyme cell distribution, its Subcellular Localization and Formation and characteristics thereof show its (Freedman that works in Protein synthesis and Secretory Pathway, 1984, TrendsBiochem.Sci.9, and this obtains support (Roth and Pierce of the direct crosslinked research of original position pp.438-41), 1987, Biochemistry, 26, pp.4179-82).Discovery (Bulleid and Freedman of the specified defect during PDI defective microsomal membrane display disulfide protein is formed, 1988, Nature, 335, pp.649-51) imply this enzyme serve as secretion property and cell surface proteins biosynthesizing during natural disulphide bonds formed catalyzer.This effect is consistent with known this enzyme catalysis characteristics in vitro; Its catalyze thiol: disulfide exchange is reacted, cause clean disulfide protein to be formed, rupture or isomerization, and can catalysis a variety of reduction unfolding protein substrate in protein folding and natural disulphide bonds form (Freedman etc., 1989, Biochem.Soc.Symp., 55, pp.167-192).The DNA of this enzyme and aminoacid sequence (Scherens, B. etc., 1991, Yeast, 7, pp.185-193 in known several species; Farquhar, R., etc., 1991, Gene, 108, pp.81-89) and have increasing information (Creighton etc., 1980, J.Mol.Biol., 142, pp.43-62 for the mechanism of action of this enzyme being purified to homogeneity from Mammals liver; Freedman etc., 1988, Biochem.Soc.Trans., 16, pp.96-9; Gilbert, 1989, Biochemistry28, pp.7298-7305; Lundstrom and Holmgren, 1990, J.Biol.Chem., 265, pp.9114-9120; Hawkins and Freedman, 1990, Biochem.J., 275, pp.335-339).Relate at present protein folding in as cell, assembling and displacement mediators the numerous protein factor in (Rothman, 1989, Cell59, pp.591-601), the unusual of PDI is in having well-defined catalytic activity.

PDI is easily separated from mammalian tissues, and the enzyme of homogeneity is the homodimer (2x57kD) (Hillson etc., 1984, MethodsEnzymol., 107, pp.281-292) with the acid pI (4.0-4.5) of characteristic.Also from wheat and this enzyme of algae Chlamydomonasreinhardii purifying (Kaska etc., 1990Biochem.J.268, pp.63-68).This activity detects in a variety of source, and in a first interim report, claims that PDI activity can detect in yeast saccharomyces cerevisiae (Williams etc., 1968, FEBSLetts., 2, pp.133-135).Report the complete amino acid sequence of many PDI recently, major part is derived from the cDNA sequence of clone; These comprise from rat (Edman etc., 1985, Nature, 317, pp.267-270), ox (Yamauchi etc., 1987, Biochem.Biophys.ReS.comm., 146, pp.1485-1492), people (Pihlajaniemi etc., 1987, EMBOJ., 6, pp.643-9), yeast (Scherens, B., etc., see above; Farquhar, R. etc., see above) and the PDI of chicken (Parkkonen etc., 1988, Biochem.J., 256, pp.1005-1011).From the protein display high degree of sequence conservative from start to finish of these invertebrate species, and be all presented in P of Rats DI sequence the several general characteristics (Edman etc. 1985, see above) first noticed.

Be identify the sequence corresponding to or be tightly coupled on PDI in the work of the function analyzed beyond disulfide formation in target.Such as, distinct evidence is had to show that PDI serves as the β subunit of tetramer α β-enzyme prolyl-4-hydroxylase, modify after the main Translation of the interior precollagen polypeptide that is newborn or that newly synthesize of this enzyme catalysis E.R. (Pihlajaniemi etc., 1987, see above; Koivu etc., 1987, J.Biol.Chem., 262, pp.6447-49)).Also evidence suggests that PDI participates in for the glycosylated system (Geetha-Habib etc. of common translation N-, 1988, Cell, 4, and propose recently mixture (Wetterau etc., 1990, J.Biol.Chem. that this enzyme participates in triglyceride level to be transferred to newborn secretion property lipoprotein pp.63-68), 265, pp.9800-7).So, PDI may be multi-functional (Freedman, 1989, Cell, 57, pp.1069-72) in the common translation and posttranslational modification of secreted protein.

The Pdi1 activity increased in bacterium, yeast and insect cell expression system can cause the secretion of the increase of the recombinant protein containing disulfide linkage.The albiglutide (ALB) that aminoacid sequence is shown in SEQIDNO:1 is made up of the DPP-4 resistance GLP-1 dimer merged to HSA.This albumen contains 8 disulfide linkage.It is possible that process LAN Pdi1 can improve SEQIDNO:1 in host cell and/or from the correct folding of host cell and secretion.

ERO1 is the gene of coding ER redox element 1 (Ero1), and this ER redox element 1 (Ero1) is the formation of protein disulfide in catalysis eukaryote endoplasmic reticulum (ER) and isomerized oxydasis reductase enzyme.(FrandAR, CuozzoJW, KaiserCA (2000). " Pathwaysforproteindisulphidebondformation " .TrendsCellBiol.10 (5): 203 – 10 and FrandAR, KaiserCA (2000). " Twopairsofconservedcysteinesarerequiredfortheoxidativeac tivityofEro1pinproteindisulfidebondformationintheendopla smicreticulum " .Mol.Biol.Cell11 (9): 2833 – 43).ERO1 from yeast saccharomyces cerevisiae has NCBI gene I/D NO.854909 and NCBI protein RefSeqNP_013576.The assumed name of ERO1 includes but not limited to: ERO1L, ERO1LB, Ero1a, Ero1b, ero-1, NCU02074.ERO1 gene is conservative in people, chimpanzee, rhesus monkey, dog, ox, mouse, rat, chicken, zebra fish, Caenorhabditis elegans, yeast saccharomyces cerevisiae, Kluyveromyces lactis (K.lactis), E.gossypii, schizosaccharomyces pombe (S.pombe), M.oryzae, Neurospora crassa (N.crassa), Arabidopis thaliana and rice.ERO1 activity includes but not limited to, the combination of flavin adenine dinucleotide, oxidoreductase activity, protein disulfide-isomerase activity and mercaptan oxidation enzymic activity.The function fragment of Ero1 and/or variant will be the polypeptide of at least one functionally active retaining wild-type Ero1.

" endoplasmic reticulum redox element " or " ERO " are the formation of protein disulfide in the endoplasmic reticulum of catalysis eukaryotic cells and isomerized oxydo-reductase.Disulfide formation is an oxidising process.After disulfide formation in protein disulphideisomerase (PDI) catalysis nascent polypeptide, PDI is reduced during thio-disulfide permutoid reaction.Need ERO to introduce the oxidation equivalent of PDI.In yeast saccharomyces cerevisiae, endoplasmic reticulum redox element is encoded by ERO1.

Show below from the gene order (SEQIDNO:7) of the ERO1 of yeast saccharomyces cerevisiae and the corresponding aminoacid sequence (SEQIDNO:8) of Ero1:

Source yeast saccharomyces cerevisiae

Source yeast saccharomyces cerevisiae

MRLRTAIATLCLTAFTSATSNNSYIATDQTQNAFNDTHFCKVDRNDHVSPSCNVTFNELNAINENIRDDLSALLKSDFFKYFRLDLYKQCSFWDANDGLCLNRACSVDVVEDWDTLPEYWQPEILGSFNNDTMKEADDSDDECKFLDQLCQTSKKPVDIEDTINYCDVNDFNGKNAVLIDLTANPERFTGYGGKQAGQIWSTIYQDNCFTIGETGESLAKDAFYRLVSGFHASIGTHLSKEYLNTKTGKWEPNLDLFMARIGNFPDRVTNMYFNYAVVAKALWKIQPYLPEFSFCDLVNKEIKNKMDNVISQLDTKIFNEDLVFANDLSLTLKDEFRSRFKNVTKIMDCVQCDRCRLWGKIQTTGYATALKILFEINDADEFTKQHIVGKLTKYELIALLQTFGRLSESIESVNMFEKMYGKRLNGSENRLSSFFQNNFFNILKEAGKSIRYTIENINSTKEGKKKTNNSQSHVFDDLKMPKAEIVPRPSNGTVNKWKKAWNTEVNNVLEAFRFIYRSYLDLPRNIWELSLMKVYKFWNKFIGVADYVSEETREPISYKLDIQ″(SEQIDNO：8)

" microorganism " means (i) prokaryotic organism, includes but not limited to, the member with subordinate: streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Bordetella (Bordetella), corynebacterium (Corynebacterium), mycobacterium (Mycobacterium), Neisseria (Neisseria), hemophilus (Haemophilus), actinomyces (Actinomycetes), streptomyces (Streptomycetes), Nocardia (Nocardia), enterobacter (Enterobacter), Yersinia (Yersinia), Fancisella, pasteurella (Pasturella), moraxella (Moraxella), acinetobacter (Acinetobacter), erysipelothrix (Erysipelothrix), Branhamella (Branhamella), Actinobacillus (Actinobacillus), Streptobacillus (Streptobacillus), listeria (Listeria), Calymmatobacterium (Calymmatobacterium), Brucella (Brucella), bacillus (Bacillus), fusobacterium (Clostridium), treponema (Treponema), Escherichia (Escherichia), salmonella (Salmonella), Klebsiella (Kleibsiella), Vibrio (Vibrio), proteus (Proteus), erwinia (Erwinia), Borrelia (Borrelia), leptospira (Leptospira), Spirillum (Spirillum), Campylobacter (Campylobacter), Shigella (Shigella), legionella (Legionella), Rhodopseudomonas (Pseudomonas), Aeromonas (Aeromonas), Dermacentroxenus (Rickettsia), chlamydiaceae (Chlamydia), Borrelia (Borrelia) and Mycoplasma (Mycoplasma), and include but not limited to further, with the member sowed or organize: group A suis, group B suis, group C suis, group D suis, group G suis, streptococcus pneumoniae (Streptococcuspneumoniae), streptococcus pyogenes (Streptococcuspyogenes), streptococcus agalactiae (Streptococcusagalactiae), streptococcus faecium (Streptococcusfaecalis), streptococcus faecalis (Streptococcusfaecium), patience suis (Streptococcusdurans), Neisseria gonorrheae (Neisseriagonorrheae), Neisseria meningitidis (Neisseriameningitidis), streptococcus aureus (Staphylococcusaureus), staphylococcus epidermidis (Staphylococcusepidermidis), corynebacterium diphtheriae (Corynebacteriumdiptheriae), Gardnerella vaginalis (Gardnerellavaginalis), Mycobacterium tuberculosis (Mycobacteriumtuberculosis), Mycobacterium bovis (Mycobacteriumbovis), mycobacterium buruli (Mycobacteriumulcerans), Mycobacterium leprae (Mycobacteriumleprae), Actinomyctesisraelii, Listeria monocytogenes (Listeriamonocytogenes), Bordetella pertussis (Bordetellapertusis), parapertussis Bo Duoshi bacillus (Bordatellaparapertusis), bordetella bronchiseptica (Bordetellabronchiseptica), intestinal bacteria (Escherichiacoli), Shigella dysenteriae (Shigelladysenteriae), hemophilus influenzae (Haemophilusinfluenzae), bacterium aegyptiacum (Haemophilusaegyptius), haemophilus parainfluenzae (Haemophilusparainfluenzae), haemophilus ducreyi (Haemophilusducreyi), Bordetella (Bordetella), salmonella typhi (Salmonellatyphi), citrobacter freundii (Citrobacterfreundii), Proteus mirabilis (Proteusmirabilis), proteus vulgaris (Proteusvulgaris), yersinia pestis (Yersiniapestis), Klebsiella pneumonia (Kleibsiellapneumoniae), serratia marcescens (Serratiamarcessens), liquefied Serratia (Serratialiquefaciens), vibrio cholerae (Vibriocholera), Shigelladysenterii, shigella flexneri (Shigellaflexneri), Pseudomonas aeruginosa (Pseudomonasaeruginosa), Francisella tularensis (Franscisellatularensis), alcaligenes abortus (Brucellaabortis), anthrax bacillus (Bacillusanthracis), Bacillus cereus (Bacilluscereus), clostridium perfringens (Clostridiumperfringens), clostridium tetani (Clostridiumtetani), Clostridium botulinum (Clostridiumbotulinum), Tyreponema pallidum (Treponemapallidum), rickettsia rickettsii (Rickettsiarickettsii) and chlamydia trachomatis (Chlamydiatrachomitis), (ii) ancient bacterium (archaeon), include but not limited to archeobacteria (Archaebacter), (iii) unicellular or fibrous eukaryote, include but not limited to, protozoon, fungi, yeast belong, the member of genus kluyveromyces (Kluveromyces) or mycocandida (Candida), and yeast saccharomyces cerevisiae, the member of the kind of Kluyveromyces lactis (Kluveromyceslactis) or Candida albicans (Candidaalbicans).

" bacterium " means (i) prokaryotic organism, includes but not limited to, the member with subordinate: streptococcus (Streptococcus), Staphylococcus (Staphylococcus), Bordetella (Bordetella), corynebacterium (Corynebacterium), mycobacterium (Mycobacterium), Neisseria (Neisseria), hemophilus (Haemophilus), actinomyces (Actinomycetes), streptomyces (Streptomycetes), Nocardia (Nocardia), enterobacter (Enterobacter), Yersinia (Yersinia), Fancisella, pasteurella (Pasturella), moraxella (Moraxella), acinetobacter (Acinetobacter), erysipelothrix (Erysipelothrix), Branhamella (Branhamella), Actinobacillus (Actinobacillus), Streptobacillus (Streptobacillus), listeria (Listeria), Calymmatobacterium (Calymmatobacterium), Brucella (Brucella), bacillus (Bacillus), fusobacterium (Clostridium), treponema (Treponema), Escherichia (Escherichia), salmonella (Salmonella), Klebsiella (Kleibsiella), Vibrio (Vibrio), proteus (Proteus), erwinia (Erwinia), Borrelia (Borrelia), leptospira (Leptospira), Spirillum (Spirillum), Campylobacter (Campylobacter), Shigella (Shigella), legionella (Legionella), Rhodopseudomonas (Pseudomonas), Aeromonas (Aeromonas), Dermacentroxenus (Rickettsia), chlamydiaceae (Chlamydia), Borrelia (Borrelia) and Mycoplasma (Mycoplasma), and include but not limited to further, with the member sowed or organize: group A suis, group B suis, group C suis, group D suis, group G suis, streptococcus pneumoniae (Streptococcuspneumoniae), streptococcus pyogenes (Streptococcuspyogenes), streptococcus agalactiae (Streptococcusagalactiae), streptococcus faecium (Streptococcusfaecalis), streptococcus faecalis (Streptococcusfaecium), patience suis (Streptococcusdurans), Neisseria gonorrheae (Neisseriagonorrheae), Neisseria meningitidis (Neisseriameningitidis), streptococcus aureus (Staphylococcusaureus), staphylococcus epidermidis (Staphylococcusepidermidis), corynebacterium diphtheriae (Corynebacteriumdiptheriae), Gardnerella vaginalis (Gardnerellavaginalis), Mycobacterium tuberculosis (Mycobacteriumtuberculosis), Mycobacterium bovis (Mycobacteriumbovis), mycobacterium buruli (Mycobacteriumulcerans), Mycobacterium leprae (Mycobacteriumleprae), Actinomyctesisraelii, Listeria monocytogenes (Listeriamonocytogenes), Bordetella pertussis (Bordetellapertusis), parapertussis Bo Duoshi bacillus (Bordatellaparapertusis), bordetella bronchiseptica (Bordetellabronchiseptica), intestinal bacteria (Escherichiacoli), Shigella dysenteriae (Shigelladysenteriae), hemophilus influenzae (Haemophilusinfluenzae), bacterium aegyptiacum (Haemophilusaegyptius), haemophilus parainfluenzae (Haemophilusparainfluenzae), haemophilus ducreyi (Haemophilusducreyi), Bordetella (Bordetella), salmonella typhi (Salmonellatyphi), citrobacter freundii (Citrobacterfreundii), Proteus mirabilis (Proteusmirabilis), proteus vulgaris (Proteusvulgaris), yersinia pestis (Yersiniapestis), Klebsiella pneumonia (Kleibsiellapneumoniae), serratia marcescens (Serratiamarcessens), liquefied Serratia (Serratialiquefaciens), vibrio cholerae (Vibriocholera), Shigelladysenterii, shigella flexneri (Shigellaflexneri), Pseudomonas aeruginosa (Pseudomonasaeruginosa), Francisella tularensis (Franscisellatularensis), alcaligenes abortus (Brucellaabortis), anthrax bacillus (Bacillusanthracis), Bacillus cereus (Bacilluscereus), clostridium perfringens (Clostridiumperfringens), clostridium tetani (Clostridiumtetani), Clostridium botulinum (Clostridiumbotulinum), Tyreponema pallidum (Treponemapallidum), rickettsia rickettsii (Rickettsiarickettsii) and chlamydia trachomatis (Chlamydiatrachomitis), (ii) ancient bacterium (archaeon), includes but not limited to archeobacteria.

As used in this article, " heterologous nucleic acid sequence " refers to insert, transform or be transfected into nucleotide sequence in interested host cell or microorganism.Heterologous nucleic acid sequence can be all or part of encoding sequence of polypeptide and/or it can comprise non-coding regulatory element, as promotor, enhanser, ribosome binding element or polyadenylation district.Heterologous nucleic acid sequence can be the not natural nucleotide sequence seeing host cell, if coding is from the nucleotide sequence of polypeptide being different from the organism of host cell, genus or kind.Or heterologous nucleic acid sequence can be that host cell gene group is natural to be had, but be inserted into, transform or be transfected in host cell with the function increasing native sequence nucleic acid or the expression of polypeptide expressed by described nucleotide sequence.Such as, wild type Saccharomyces cerevisiae can contain the nucleotide sequence of encoding wild type Kex2p, but the heterologous nucleic acids of encoding wild type Kex2p can be transformed in described yeast saccharomyces cerevisiae to improve the Kex2p production of described host cell.Similarly, wild type Saccharomyces cerevisiae can contain the nucleotide sequence of encoding wild type Kex2p, but the heterologous nucleic acids of the encoded K ex2p from different organism can be inserted in described yeast saccharomyces cerevisiae to improve the Kex2p production of described host cell.The host cell containing following heterologous nucleic acid sequence is also contained in the present invention, and it is variant and/or the fragment of the wild-type nucleic acid of host cell from same species.

As used in this article, " recombinant polypeptide " and grammatical variants thereof refer to not by the object host cell be converted or the natural synthesis of microorganism, but introduce the polypeptide in described host cell or microorganism by recombinant DNA.Such as, yeast saccharomyces cerevisiae can serve as host cell and not be present in unconverted or without the human serum albumin in transfection yeast saccharomyces cerevisiae for expressing.Recombinant polypeptide can comprise modified to assist isolated polypeptide.

As used in this article, " affinity tag " refers to any module of combining with molecule, and it can give the selective affinity of described molecule to another material or molecule.Such as, affinity tag can be used for the purifying of assisting molecule, it is by providing the selective affinity of the packing material of coupled columns for molecule.The non-limitative example of affinity tag is his label.

Nucleic acid is " being operatively connected " when it is placed in the functional relationship with another nucleotide sequence.Such as, if the DNA of presequence (presequence) or secretion leader sequence is with protein expression before participating in polypeptide and secreting, then it is operably connected the DNA of coded polypeptide; If promotor or enhanser affect transcribing of sequence, then it is operably connected encoding sequence; If or ribosome bind site is orientated as and made to promote translation, then it is operably connected encoding sequence.Usually, " being operably connected " means that the DNA sequence dna be joined together is continuous print, and secreting the situation of leader sequence, being continuous print and meeting reading frame.But enhanser needs not to be continuous print.Connection is by having connected at restriction site place easily.If there is no such site, conveniently way uses synthetic oligonucleotide adapter or joint.

As used in this article, " results " cell refers to from cell culture collecting cell.Can by such as centrifugal or filter between harvesting time concentrating cells so that it is separated from nutrient solution.Harvested cell can comprise further lysing cell with obtain material in born of the same parents (as but be not limited to polypeptide and polynucleotide) step.Those skilled in the art should understand can discharge some cell material from cell in the training period, includes but not limited to, the polypeptide of heterogenous expression.So, interested product (such as recombinant expressed polypeptide) can remain in nutrient solution after harvested cell.

Further, the method for wherein recombinant dna construct encodes selectable marker is provided.This kind of mark of selecting provides positive or negative to select.Also provide following methods, it comprises expressing described can select mark and the amount by the mark selected selecting at least one first transformant of step to produce be compared with the amount by the mark selected selecting at least one second transformant of step to produce, the mark selected that wherein said first and second transformants generations are identical.As understood in the art, mark can be selected to include but not limited to, Tetrahydrofolate dehydrogenase (dhfr), beta-galactosidase enzymes, fluorescin, the secreted form of placental alkaline phosphatase, beta-glucuronidase, yeast can select mark LEU2 and URA3, apoptosis resistance gene and antisense oligonucleotide, and give the antibiotics resistance gene of the ability grown when there is following microbiotic, comprise Liu Suanyan NEOMYCIN SULPHATE (neo), kantlex, Geneticin (geneticin), hygromycin B, tetracycline, zeocin, blasticidin, nourseothricin (nourseothricin), bialaphos (bialaphos), phleomycin (phleomycin) and penbritin.As also understood in this area, carrying out sorting cells by multiple means, including but not limited to, visual inspection or cell sorter are as BDFACSAria, and it can detect the expression can selecting mark.

As understood in this area, term " wild-type " refers to be present in does not have host cell in the natural population of genetic modification or polypeptide or polynucleotide sequence.Such as, " wild-type host cells " refers to carry out in the genome of host cell or not modified host cell strains before there is any genetic modification.

As used in this article, " titre " or " titre productive rate " refers to the concentration of product (such as recombinant expressed polypeptide) in solution (such as nutrient solution or lysis mixture or damping fluid) and is typically expressed as mg/L or g/L.The increase of titre productive rate can refer to definitely or relatively increasing of the production concentration produced under the condition group of two restrictions.

" GLP-1 " means reinforcement insulin secretion or increases any hormone of insulin level in another manner as used in this article.An example of GLP-1 is GLP-1.GLP-1 is the incretin secreted by intestines L cellular response ingestion of food.In the individuality of health, GLP-1 plays an important role in adjustment level of postprandial blood sugar, and its glucose dependent insulin by stimulating pancreas is secreted, and produces the glucose absorption increased in outer peripheral areas.GLP-1 also glucagon suppression secretion, causes the hepatic glucose reduced to export.In addition, GLP-1 postpones the gastric emptying time and delays small bowel motility, causes the food absorption postponed.GLP-1 by stimulate the gene that relates in glucose dependent insulin secretion transcribe and by the beta cell ability (competence) that promotes beta cell neogenesis to promote to continue, (Meier, waits Biodrugs2003; 17 (2): 93-102).

" GLP-1 is active " means one or more activity of naturally occurring people GLP-1 as used in this article, include but not limited to, reduce blood and/or plasma glucose, stimulate glucose dependent insulin secretion or improve insulin level in another manner, glucagon suppression is secreted, and reduces fructosamine, increases glucose to the delivery of brain and metabolism, postpone stomach emptying, and promote beta cell ability and/or new life.These are active and directly or indirectly can be caused by the composition or GLP-1 agonist with GLP-1 activity with any one in active other the relevant activity of GLP-1.Such as, the composition with GLP-1 activity can directly or indirectly stimulate glucose dependency to stimulate Regular Insulin to produce the plasma glucose levels that then may indirectly reduce in Mammals.

" incretin stand-in " are the compounds can strengthened insulin secretion or improve insulin level in another manner as used in this article.Incretin stand-in and can be induced full in Mammals moderate stimulation insulin secretion, increase beta cell neogenesis, suppression beta apoptosis, glucagon suppression secretion, delay stomach re-scheduling.Incretin stand-in can include but not limited to, have any polypeptide of GLP-1 activity, include but not limited to, exendin3 and exendin4, comprise its any fragment and/or variant and/or conjugate.

" domain antibodies " or " dAb " can be considered as identical with " single variable domain ", and it can conjugated antigen.Single variable domain can be people's antibody variable domains, but also comprises from other species as rodentine single antibody variable domains (such as disclosed in WO00/29004), nurse shark and camel class V _hHdAb.Camel class V _hHbe be derived from camel (camel), Llama (llama), alpaca (alpaca), dromedary camel (dromedary) and llama (guanaco) the immunoglobulin (Ig) list variable domain polypeptide at interior species, it produces the natural heavy chain antibody not having light chain.This kind of V _hHterritory can according to the standard technique humanization that can obtain in this area, and this class field is regarded as " domain antibodies ".V as used in this article _hcomprise camel class V _hHterritory.

Phrase " single variable domain " refers to antigen-binding proteins variable domain (the such as V of specific binding antigen or epi-position _h, V _hH, V _l), this combination is independent of different variable regions or territory.

Term as used in this article " antigen-binding proteins " refers to can the antibody of conjugated antigen, antibody fragment and other protein constructs, as territory, but are not limited to, variable domain and domain antibodies.

As used in this article, " amount of reduction " and the grammatical variants thereof of the enzyme compared in genetically modified host cell or its fragment or enzymic activity refer to a kind of genetically modified host cell, and it produces less at least one enzyme or shows less at least one enzymic activity compared with not genetically modified host cell.Usually, the wild type strain before the genetic modification of the enzymic activity produced by genetically modified host cell and same species is compared.But, relatively can also genetically modified host and from this genus but for different plant species or bacterial strain wild-type host between or the bacterial strain genetically modified with another kind compare.The reduction of at least one enzyme or enzymic activity also comprises eliminates at least one enzyme or enzymic activity completely, does not have a kind of resulting from genetically modified host cell and/or described at least one enzyme not have one to be functional or display activity in wherein said at least one enzyme.Also included within this definition is at least one enzymic activity of reducing amount.That is, there is the enzyme exceeding a kind of activity and can maintain the amount of the first activity and reduce the second active of same enzyme simultaneously.

As used in this article, in genetically modified host cell, " amount of increase " of enzyme or its fragment or enzymic activity and grammatical variants thereof refer to a kind of genetically modified host cell, and it produces more at least one enzyme or shows more at least one enzymic activity compared with not genetically modified host cell.Usually, the wild type strain before the genetic modification of the enzymic activity produced by genetically modified host cell and same species is compared.But, relatively can also genetically modified host and from this genus but for different plant species or bacterial strain wild-type host between or the bacterial strain genetically modified with another kind compare.Also included within this definition is at least one enzymic activity of increasing amount.That is, there is the enzyme exceeding a kind of activity and can maintain the amount of the first activity and increase the second active of same enzyme simultaneously.In addition, this term comprises except the enzyme amount that host cell produces, and increases enzymic activity.Such as, genetically modified host cell can produce and the enzyme of the same or similar amount produced by wild-type host cells or its fragment and/or variant, as passed through quality (mass) or measurement amount, but measurable increase can be had compared to wild-type in the amount of at least one functionally active of described enzyme.

As used in this article, term " stringent condition " and " stringent hybridization condition " mean hybridization only has at least 70% and at least 80% between sequence, but occurs when at least 95% identity.An example of stringent hybridization condition is comprising Overnight incubation in following solution at 42 DEG C: 50% methane amide, 5xSSC (150mMNaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x steps on Hart (Denhardt) solution, 10% T 500, and 20 micrograms/ml sex change, shearing salmon sperm DNA, then in 0.1xSSC at about 65 DEG C of cleaning filters.Hybridization and cleaning condition are known and are illustrated in Sambrook, Deng, MolecularCloning:ALaboratoryManual, SecondEdition, ColdSpringHarbor, N.Y., (1989), particularly wherein Chapter 11, is incorporated to entire disclosure herein thus by carrying stating.

As used in this article, " genetic modification " or " genetically modified " refers to any suppression to one or more base of cell DNA sequence or fragment, replacement, deletion and/or insertion.This kind of genetic modification can external (directly be separated DNA on) or original position obtain, such as, by genetic engineering techniques or by cell is exposed to mutagenic compound.Mutagenic compound comprise such as, physical factor as energy-ray (X-ray, gamma-radiation, UV etc.) or the chemical reagent that can react with the different functional groups of DNA, as alkylating agent (EMS, NQO etc.), double alkylation agent, intercalator etc.Genetic modification also by genetic disruption, such as, obtains (Meth.Enzymol.194:281-301 (1991)) according to disclosed methods such as Rothstein.According to the method, part or all of gene is replaced with external modified pattern via homologous recombination.Genetic modification can also be inserted, as the acquisition such as transposon, phage by any sudden change on DNA sequence dna.Further, " genetically modified " can refer to gene or the polypeptide of coded polypeptide as used in this article, and it has at least one disappearance respectively, replaces or suppress in nucleic acid or amino acid.Such as, wherein the polypeptide that is substituted from wild-type form of at least one amino acid is genetically modified by being considered as.

Reverse by cell mechanism or weaken genetic modification.Or sudden change can be non-reply or non-seepage." leakage sudden change " comprises the sudden change causing wild-type funtion part but not complete deactivation.

The genetic modification that host cell of the present invention carries can be arranged in the coding region of cell DNA sequence and/or affect the region of genetic expression.Therefore, impact is generally related to the gene product of proteolysis and/or glycosylated protein and/or enzyme or the regulation and control of gene product or promotion by modification of the present invention.It may be due to structure and/or conformational change that the ability of the polypeptide that cell protein enzymolysis cutting of the present invention and/or glycosylated heterologous are expressed reduces, from the generation of one or more enzymes of biological characteristics with change, from the generation lacking one or more enzymes described, or produce with low-level from one or more enzymes.

Genetic modification of the present invention also affects regulation and control or the promotion of the protein of any one functionally active relating to Kex2p, Pdi1 and ero1 described herein and/or the gene product of enzyme or gene product.The cell of the present invention correctly ability of increase that is folding and that secrete recombinant expressed polypeptide may be that enzyme owing to relating to these processes has the biological characteristics of change or produces with high level.

In one aspect of the invention, genetically modified host cell is provided, it comprises the polynucleotide that at least one that coding kills and wounds expression (KEX) proteolytic enzyme (Kex2p) or have its fragment of at least one Kex2p protease function activity and/or variant is separated, and the polynucleotide that at least one of proteins encoded disulphide isomerase (Pdi1) or its fragment and/or variant with at least one Pdi functionally active is separated.Genetically modified host cell of the present invention comprises following genetically modified host cell, and it comprises coding endoplasmic reticulum redox element (ero1) or has the polynucleotide of its fragment of at least one ERO functionally active and/or at least one separation of variant.

Genetically modified host cell of the present invention also comprises following genetically modified host cell, when described genetically modified host cell is cultivated in culture, compared to the second host cell, the at least one gene product of the polynucleotide that it is expressed or process LAN at least one is separated, the polynucleotide encoding of described separation is selected from the protein of lower group and/or has its variant of at least one functionally active of described protein: Kex2p, Pdi1 and Ero1, wherein said second host cell is not expressed or process LAN is not selected from KEX, the at least one gene product of PDI and ERO.The present invention also comprises following genetically modified host cell, when described genetically modified host cell is cultivated in culture, compared to the second host cell, its process LAN at least two kinds is selected from the protein of lower group or has its fragment of at least one functionally active and/or the variant of described protein: Kex2p, Pdi1 and Ero1, and wherein said second host cell belongs to same species and to cultivate under same culture conditions but process LAN is not selected from least two kinds of gene products of KEX, PDI and ERO.In some cases, second host cell can have genetic modification, but do not have the genetic modification of at least one gene product allowing it is expressed or process LAN at least one is separated polynucleotide, the polynucleotide encoding of described separation is selected from the protein of lower group and/or has its variant of at least one functionally active of described protein: Kex2p, Pdi1 or Ero1.In some cases, the second host cell can be the wild-type cell (namely without genetic modification) with modified host cell same species.In some cases, except comprising the nucleic acid of its variant of the protein of encoding and being selected from lower group and/or at least one functionally active with described protein, the second host can containing all genetic modifications identical with genetically modified host cell.The present invention is also contained through genetically modified host cell of expressing with the endogenous polypeptide improving the gene contained from host cell, and described polypeptide includes but not limited to: Kex2p, Pdi1 and Ero1.

In another aspect of the present invention, there is provided genetically modified host cell, it comprises the following genetic modification of at least one further: pep4 proteolytic enzyme knocks out, ubc4 and/or ubc5 lower compared to wild-type host cells active, yps1 knocks out, hsp150 knocks out and knocks out with pmt1.Find that these genetic modifications increase recombinant human serum white protein secretion capacity and reduce undesired posttranslational modification.

The yeast strain used in the production of albumin fusion proteins includes but not limited to D88, DXY1 and BXP10.D88 [leu2-3, leu2-122, can1, pra1, ubc4] is that the derivative of parent strain AH22his.sup.+ is (also referred to as DB1; See Biotechnology8:42-46 (1990) such as such as Sleep).This bacterial strain contains leu2 sudden change, and it allows the auxotrophy Sexual behavior mode based on the plasmid of 2 microns containing LEU2 gene.D88 also show glucose superfluous time the derepressing of PRB1.PRB1 promotor is normally controlled by two check points of monitoring glucose level and growth phase.When glucose exhausts and enter stationary phase, in wild-type yeast, promotor is activated.Bacterial strain D88 shows checking but maintaining induction when entering stationary phase by glucose.PRA1 genes encoding is positioned the yeast vacuole proteolytic enzyme YscA endo-protease A in ER.UBC4 gene is in ubiquitination pathway and relate to target the life-span short and protein of exception to carry out the degraded of ubiquitin dependency.Find that separation that this ubc4 suddenlys change increases the copy number of expression plasmid in cell and causes increasing (see such as International Publication text No.WO99/00504, stating complete being incorporated herein by carrying) from the level of the expectation protein of plasmid expression.

The derivative DXY1 of D88 has following genotype: [leu2-3, leu2-122, can1, pra1, ubc4, ura3:yap3].Except the sudden change be separated in D88, this bacterial strain also has knocking out of YAP3 proteolytic enzyme.This proteolytic enzyme causes the cutting of the two alkaline residue (RR, RK, KR, KK) of great majority but also can promote the cutting at single base residue place in protein.The higher level (see such as U.S. Patent No. 5,965,386 and Kerry-Williams etc., Yeast14:161-169 (1998), states complete being incorporated herein by carrying thus) that the separation that this yap3 suddenlys change causes total length HAS to produce.

BXP10 has following genotype: leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3.Except the sudden change be separated in DXY1, this bacterial strain also has knocking out of PMT1 gene and HSP150 gene.PMT1 gene is the member of the evolution conservative family of polyterpene base phosphoric acid-D-MANNOSE albumen O-mannose transferase (Pmt).The Transmembrane Topology of Pmt1p shows that it is effective endoplasmic reticulum AQP-CHIP in the glycosylation of O-connection.The glycosylation (see such as International Publication text No.WO00/44772, stating complete being incorporated herein by carrying thus) that this mutation effect connects in the O-reducing/eliminate HAS fusions.Research is disclosed Hsp150 albumen and is separated from rHA poor efficiency by ion exchange chromatography.Sudden change in HSP150 gene eliminates potential pollutent, this pollutent be proved to be by standard purification techniques be difficult to remove.See such as U.S. Patent No. 5,783,423, state complete being incorporated herein by carrying thus.

Genetically modified host cell of the present invention includes but not limited to, fungal cell, yeast cell and mammalian cell.Genetically modified host cell of the present invention includes but not limited to: yeast belong (Saccharomyces), genus kluyveromyces (Kluyveromyces), mycocandida (Candida), Pichia (Pichia), Schizosaccharomyces (Schizosaccharomyces), Hansenula (Hansenula), Ke Leke yeast (Kloeckera), permitted prosperous yeast belong (Schwanniomyces) and Ye Shi yeast belong (Yarrowia).Genetically modified host cell of the present invention also includes but not limited to yeast saccharomyces cerevisiae.

Genetically modified host cell of the present invention also can comprise at least one polynucleotide of encoding recombinant polypeptide.The polynucleotide can expressing at least one heterologous polypeptide include but not limited to, carrier, the DNA be transformed in host cell gene group, virus or viral part and/or plasmid.The polynucleotide of expressing heterologous polypeptide can be transformed in the genome of host cell and/or can be the part of expression vector and/or episome expression system.

More of the present invention in, the nucleic acid of encoding recombinant polypeptide is included in plasmid.In other respects, the nucleic acid of encoding recombinant polypeptide is transformed in the genome of host cell of the present invention.

As understood in this area, by several diverse ways, DNA is transformed in host cell.In yeast, what DNA can be used to shift anyly facilitates method, as electroporation, lithium chloride method, lithium acetate method or spheroplast method.In order to produce the blast resistance of applicable high density fermentation, expect DNA to be incorporated in host chromosome.Use technology as known in the art, integrate via homologous recombination.Such as, provide the flanking sequence with host organisms sequence homology can to the DNA that can express at least one heterologous protein.By this way, integrate the restriction site occurred in host genome, and do not destroy the gene of expectation or necessity.In addition/or the DNA that can express at least one heterologous protein is incorporated in the site of undesired gene in host chromosome, thus realize destruction or the disappearance of the expression of this gene or this gene product.

The expression of the increase of gene product or process LAN are by the DNA of expressing gene product can be incorporated into additional copy in host chromosome and realize.In addition/or the DNA of encodes gene product can be operably connected to strong promoter, and complete expression cassette can be incorporated into the site limited in host chromosome.Such as, by being operably connected to the encoded K ex2p of strong promoter (such as PGK1 promotor), the DNA of Pdi1 or Ero1 is incorporated into the process LAN allowing corresponding gene product in the site of NTS2-2.In other embodiments, via in karyomit(e), plasmid, retroviral vector or random integration to host genome, DNA can be introduced host.

Genetically modified host cell of the present invention comprises following genetically modified host cell, and the polynucleotide that wherein encode kex proteolytic enzyme or at least one with its fragment of at least one Kex2p functionally active and/or variant are separated are operably connected at least one promotor being selected from lower group: TEF1, PRB1ADH1, ADH2, PYK1, PGK1, ENO, GAL1.10.7, GALS, MET25, CUP1, PHO5, tetO-CYC1, CaMV, HXT6, HXT7 and ARE.Promotor is suitably PGK1.Genetically modified host cell of the present invention also comprises following genetically modified host cell, and the polynucleotide that wherein encode PDI or at least one with its fragment of at least one Pdi functionally active and/or variant are separated are operably connected at least one promotor being selected from lower group: TEF1, PRB1ADH1, ADH2, PYK1, PGK1, ENO, GAL1.10.7, GALS, MET25, CUP1, PHO5, tetO-CYC1, CaMV, HXT6, HXT7 and ARE.Promotor is suitably PGK1.In addition, genetically modified host cell of the present invention comprises following genetically modified host cell, and the polynucleotide that wherein encode ERO or at least one with its fragment of at least one ERO functionally active and/or variant are separated are operably connected at least one promotor being selected from lower group: TEF1, PRB1ADH1, ADH2, PYK1, PGK1, ENO, GAL1.10.7, GALS, MET25, CUP1, PHO5, tetO-CYC1, CaMV, HXT6, HXT7 and ARE.Promotor is suitably PGK1.

In yet another aspect, the recombinant polypeptide of expressing in genetically modified host cell of the present invention has at least one disulfide linkage.In some respects, described recombinant polypeptide is albumin fusion proteins.In some respects, described recombinant polypeptide comprises at least one therapeutical peptide with GLP-1 activity and puts together in white protein.

In some respects, at least one fragment of GLP-1 and variant comprise GLP-1 (7-36 (A8G)) and genetic fusion in human serum albumin.In still another embodiment, the GLP-1 molecule of polypeptide of the present invention comprise N or the C-terminal being blended in human serum albumin or its variant 1,2,3,4,5 or more series connection orientation and/or its fragment and/or variant.Other embodiments have this kind of A8G polypeptide of N or the C-terminal being blended in white protein or its variant.GLP-1 (7-36) (A8G) fragment and/or the variant fusion of two series connection orientations comprise SEQIDNO:1 in an example of the N-terminal of human serum albumin, and it is presented in Fig. 3.In yet another aspect, at least one fragment of GLP-1 and variant comprise series connection and genetic fusion at least two GLP-1 (7-36 (A8G)) of human serum albumin.At least two GLP-1 (7-36 (A8G)) can at the N-terminal genetic fusion of human serum albumin.The polypeptide that at least one has GLP-1 activity can comprise SEQIDNO:1.

The variant of GLP-1 (7-37) can be expressed as such as Glu ²²-GLP-1 (7-37) OH, the glycine that its instruction is wherein just being common in GLP-1 (7-37) OH the 22nd is by GLP-1 variant that L-glutamic acid is replaced; Val ⁸-Glu ²²the L-Ala that-GLP-1 (7-37) OH instruction is wherein just being common in GLP-1 (7-37) OH the 8th and be just common in the 22nd glycine respectively by the GLP-1 compound of α-amino-isovaleric acid and L-glutamic acid replacement.The example of GLP-1 variant includes but not limited to,

The variant of GLP-1 can also include but not limited to, one or more amino acid side groups has GLP-1 or the GLP-1 fragment of chemically modified.Chemically modified includes but not limited to, adds chemical module, creates new key, and removing chemical module.The modification at amino acid side groups place includes but not limited to, the acidylate of Methionin-epsilon-amino group, the N-alkylation of arginine, Histidine or Methionin, the alkylation of L-glutamic acid or aspartic carboxylic acid groups, and the desamidization of glutamine or l-asparagine.The modification of terminal amino group includes but not limited to, de--amino, N-low alkyl group (alkyl), N-be two-and low alkyl group and the modification of N-acyl group.The modification of terminal carboxyl groups includes but not limited to, acid amides, lower alkyl, dialkyl amide and lower alkyl esters are modified.In addition, by having the known protectiveness group of the protein chemistry man of ordinary skill to protect one or more side-chain radicals or end group.

GLP-1 fragment or variant also can comprise the N-terminal of GLP-1 (7-37) OH and/or the polypeptide at C-terminal place that wherein one or more amino acid have added described fragment or variant to.The amino acid that wherein amino acid has been added in the GLP-1 at N-terminal or C-terminal place is indicated by the numbering identical with the corresponding amino acid in GLP-1 (7-37) OH.Such as, by adding the N-terminal amino acid of the GLP-1 compound that two amino acid obtain to the N-terminal of GLP-1 (7-37) OH in position 5; And by C-terminal amino acid from the GLP-1 compound that amino acid obtains to the C-terminal of GLP-1 (7-37) OH that add in position 38.So, in these two kinds of GLP-1 compounds, position 12 is occupied by phenylalanine, and position 22 is occupied by glycine, as in GLP-1 (7-37) OH.Amino acid is added to the amino acid/11-6 of the GLP-1 of N-terminal can or conservative replacement identical with the amino acid of the corresponding position of GLP-1 (1-37) OH.Amino acid is added to the amino acid 38-45 of the GLP-1 of C-terminal can or conservative replacement identical with the amino acid of the corresponding position of hyperglycemic-glycogenolytic factor or exendin-4.

In yet another aspect, at least one polypeptide with GLP-1 activity comprises at least one people GLP-1 fragment and/or variant that are blended in human serum albumin.In yet another aspect, at least one fragment of GLP-1 and variant comprise GLP-1 (7-36 (A8G)).At least one fragment of described GLP-1 and variant genetic are blended in human serum albumin.In yet another aspect, recombinant polypeptide of the present invention comprise series connection and genetic fusion at least two GLP-1 (7-36 (A8G)) of human serum albumin.Described two GLP-1 (7-36 (A8G)) are at the N-terminal place genetic fusion of human serum albumin.In some cases, recombinant polypeptide comprises SEQIDNO:1.

In one embodiment of the invention, recombinant polypeptide comprises the polypeptide with listed polypeptide in SEQIDNO:1 with 99% sequence iden, or has the polypeptide of aminoacid sequence of the SEQIDNO:1 in C-terminal and/or N-terminal brachymemma.In one aspect, recombinant polypeptide has GLP-1 activity.In one aspect, compared to SEQIDNO:1, polypeptide in N-terminal brachymemma 1,2,3,4,5,6,7,8,9,10 amino acid, or polypeptide has 99% sequence iden with SEQIDNO:1 in whole sequence.In one aspect, compared to SEQIDNO:1, recombinant polypeptide in C-terminal brachymemma 1,2,3,4,5,6,7,8,9,10 amino acid, or polypeptide has 99% sequence iden with SEQIDNO:1 in whole sequence.

In a further embodiment, at least one recombinant polypeptide of expressing in host cell of the present invention comprises following one or more: at least one antigen-binding proteins, at least one list variable domain and/or at least one domain antibodies.The polypeptide comprising at least one antigen binding domain also can comprise at least one polypeptide and/or peptide receptor agonist and/or antagonist.In some cases, described polypeptide agonist can be GLP-1 receptor stimulant.As understood in this area, exceeding a kind of recombinant polypeptide can at same cells.Such as, the recombinant polypeptide with GLP-1 activity can at the identical cells with antigen-binding proteins.The polypeptide with GLP-1 activity can be expressed from the polynucleotide identical with antigen-binding proteins, and these polynucleotide are operably connected to expresses necessary nucleotide sequence.Or and such as, the polypeptide with GLP-1 activity can independent of the second recombinant polypeptide as antigen-binding proteins be operably connected to and expresses required different polynucleotide sequences from same episomal DNA or genome and express, or expresses from the DNA sequence dna be positioned at separately carrier.

Genetically modified host cell is also provided, the polynucleotide that its at least one comprising its fragment and/or variant that coding kills and wounds expression (KEX) proteolytic enzyme (Kex2p) or has at least one Kex2p functionally active is separated, the polynucleotide that proteins encoded disulphide isomerase (Pdi1) or at least one with its fragment of at least one Pdi functionally active and/or variant are separated, and coding endoplasmic reticulum redox element (Ero1) or there is its fragment of at least one ERO functionally active and/or at least one heterologous nucleic acid sequence of variant.Genetically modified host cell of the present invention comprises at least one nucleic acid of encoding recombinant polypeptide.In another aspect of the present invention, when cultivating in culture, with belong to same species and have identical genetic modification but do not comprise the polynucleotide sequence that at least one that coding kills and wounds its fragment and/or variant expressing (KEX) Proteinase K ex2p or have at least one KEX functionally active is separated, the polynucleotide that proteins encoded disulphide isomerase (Pdi1) or at least one with its fragment of at least one Pdi functionally active and/or variant are separated, the host cell of the polynucleotide be separated with at least one of coding endoplasmic reticulum redox element (Ero1) or its fragment and/or variant with at least one Ero1 functionally active is compared, described genetically modified host cell improves the expression of described recombinant polypeptide.In some cases, genetically modified host cell is yeast saccharomyces cerevisiae.

In yet another aspect, the recombinant polypeptide of expressing in genetically modified host cell of the present invention comprises and has at least 95%, 96% with SEQIDNO:1, the aminoacid sequence of 97%, 98%, 99% or 100% sequence iden.

In yet another aspect, provide the method producing recombinant polypeptide, comprise and cultivate genetically modified host cell of the present invention.In other respects, described method also comprises and reclaims described recombinant polypeptide from substratum.In other respects, the recombinant polypeptide prepared by described method is provided.In yet another aspect, the recombinant polypeptide prepared by method of the present invention comprises the aminoacid sequence with SEQIDNO:1 with 99% sequence iden.In other respects, described recombinant polypeptide comprises aminoacid sequence listed in SEQIDNO:1.On the other hand, recombinant polypeptide comprises leader sequence.In one aspect, described leader sequence is modified KEX leader sequence, and it comprises aminoacid sequence listed in SEQIDNO:10.As understood in this area, host cell and growth conditions can affect the end product of the recombinant protein produced by host cell.Such as, posttranslational modification can be realized by host cell species and growth conditions.These posttranslational modifications (include but not limited to glycosylation and methylate) of recombinant protein can realize following aspect, as but be not limited to, the protein folding of the recombinant protein produced by described host cell and protein active or effect.

In another aspect of the invention, provide a kind of pharmaceutical composition, it comprises the recombinant polypeptide prepared by the inventive method.The method for the treatment of the patient having this to need also is provided, comprises the described pharmaceutical composition of administering therapeutic significant quantity.In some cases, patient suffers from and is selected from following disease or situation: type i diabetes, type ii diabetes, glucose intolerance, hyperglycemia, Alzheimer's, obesity, cardiovascular disorder, congestive heart failure and retinopathy.

As used in this article, " therapeutical peptide " refers to have one or more treatment and/or biologic activity, particularly can be used for the protein of at least one biologic activity treating, prevent or improve disease, polypeptide, antibody, peptide or its fragment or variant.The therapeutical peptide that the present invention is contained includes but not limited to, protein, polypeptide, peptide, antibody and biotechnological formulation.(term peptide, proteins and peptides can exchange use in this article).A non-packet capacitive list of the biologic activity that therapeutical peptide can have comprises, any GLP-1 described herein is active, strengthen immunne response, promote that blood vessel occurs, suppress blood vessel to occur, endocrine regulation function, regulate hemopoietic function, to excite nerve growth, strengthen immunne response or Immunosuppression response.

As used in this article, " patient " is the animal suffering from disease, situation or illness, and being preferably Mammals, is most preferably people.

As used in this article, " treatment significant quantity " refers in treatment, prevents or improve in disease, situation or illness effectively to measure.To treat, suppressing or prevent to determine by standard clinical techniques with the amount of effective pharmaceutical composition of the present invention in the unconventionality expression of therapeutical peptide and/or active relevant disease or illness.In addition, vitro assay can be optionally adopted to help identify best dosage range.The correct dose that will adopt in the formulation also will depend on the severity of administration route and disease or illness, and should decide according to the situation of the judgement of practitioner and each patient.Effective dose can from the dosage-response curve extrapolation being derived from external or animal model test macro.

As used in this article, " pharmaceutical composition " comprises therapeutical peptide and pharmaceutically acceptable carrier.In a specific embodiment, term " pharmacy is acceptable " means to be ratified by the regulator of federal government or state government, or lists in American Pharmacopeia or other It is generally accepted for animal, more specifically for the pharmacopeia of people.Term " carrier " refers to thinner, adjuvant, vehicle or the vehicle used together with therapeutant.This type of pharmaceutical carrier can be aseptic liquid, and such as water and oil, comprise oil, and animal, plant or synthesis are originated, such as peanut oil, soybean oil, mineral oil, sesame oil etc.When intravenously drug administration composition, water is preferred carrier.Salt brine solution and aqueous dextrose and glycerine solution also can be used as liquid vehicle, especially for Injectable solution.The drug excipient be applicable to comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, skim-milk, glycerine, propylene, glycol, water, ethanol etc.If you want it, described composition also can contain a small amount of wetting agent or emulsifying agent, or pH buffer reagent.These compositions can take the form of solution, suspension agent, emulsion, tablet, pill, capsule, pulvis, sustained release preparation etc.Described composition can be formulated as suppository, uses traditional tackiness agent and carrier such as triglyceride level.Oral preparations can comprise carrier such as pharmaceutical grades of mannitol, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, the magnesiumcarbonate etc. of standard.The example of the pharmaceutical carrier be applicable to is described in " Remington'sPharmaceuticalSciences " of E.W.Martin.This based composition by containing the treatment compound of significant quantity or therapeutical peptide (the being preferably purified form) carrier together with sufficient quantity, thus provides the suitable form used for patient.Preparation should be applicable to mode of administration.

As understood in this area, " in culture cultivate/growth " and grammatical variants refers to host cell inoculating nutrient medium and Incubate cells culture (usually under the condition for concrete host cell growth the best or standard) to allow Growth of Cells and/or division.As understood in this area, the enzymic activity of one or more enzymes produced by the host cell in cultivating can be subject to the impact of the growth conditions of culture.Such as, the proteolytic activity of the proteolytic enzyme produced by the host cell in cultivation reduces by changing one or more following conditions: pH, dissolved oxygen, temperature, osmolarity (osmolarity), one or more nutrient media componentses, specific protein enzyme inhibitors, growth time and/or speed, cell concn, cultivation time length and/or glucose feed rate (such as batch feed).Adding complex proteins hydrolysate to culture may be especially effective for arrestin enzymolysis.And, in the training period can to make the mode of maximum effect to change condition during one or more specified time.Similarly, the glycosylation of the protein produced in culture can by the impact of similar factor.Therefore, for reducing or the growth conditions of the enzymic activity (as proteolysis or glycosylation activity) that increases host cell in culture optimize by one or more nonrestrictive factors that adjustment is listed above.

Further, as understood in this area, in host cell, the generation of heterologous protein and/or recombinant protein increases by controlling many same factors mentioned above.In addition, add the factor (include but not limited to, add rapamycin (rapamycin) to growth medium) increasing vector copies and also can increase production.Other factors that can increase production include but not limited to, one or more chaperones of coexpression, as protein disulphideisomerase (PDI).In addition, oxyphorase (HB) can in host cell, coexpression be to strengthen the oxygen availability of oxidative metabolism together with at least one heterologous polypeptide, and so increase polypeptide produces.

In yet another aspect, leader sequence is comprised from the recombinant polypeptide of genetically modified host cell expression of the present invention.In some respects, described leader sequence is KEX2 leader sequence or modified KEX2 leader sequence.

Wild-type KEX leader sequence is hereafter being shown as SEQIDNO:9.

MetLysTrpValSerPheIleSerLeuLeuPheLeuPheSerSerAraTyrSerArgSerLeuAspLysArg(SEQIDNO：9)

In some cases, the modified KEX leader sequence being shown as SEQIDNO:10 is used.

MetLysTrpValSerPheIleSerLeuLeuPheLeuPheSerSerAlaTyrSerGlySerLeuAspLysArg(SEQIDNO：10)

In some cases, KEX leader sequence can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence iden with SEQIDNO:9 in whole sequence.

Secretory leader sequence can be comprised from the heterologous protein of host cell secretes or recombinant protein at production period.Leader sequence can be passed through to be modified to improve secretion, improves overall generation and the recovery of heterogenous expression protein thus; Different leader sequences such as from multiple secretory protein can be operably connected to heterologous protein and the expression of assessment enhancing.Or given leader sequence is modified by site-directed mutagenesis, or modified by the leader sequence variant that the qualification of combinatorial library way improves.Can find that the chimeric leader sequence comprised from the region of two or more leading peptides improves heterologous protein expression level.

Embodiment

Following examples are exemplified with multiple non-limiting aspect of the present invention.

Embodiment 1 – process LAN KEX2, the Wine brewing yeast strain of PDI1 and ERO1

Be used in Nottingham, the yeast host expression system (yeast saccharomyces cerevisiae BXP10) that the DeltaBiotechnologyLtd. (Delta) of UK develops builds the Wine brewing yeast strain of process LAN KEX2, PDI1 and ERO1.BXP10 is derived from Wine brewing yeast strain AH22, and it obtains from ATCC and is derived from s288c.The structure of BXP10 relates to a series of random mutagenesis and targeting specific gene destroys increase recombinant human serum white protein (rHSA) secretion capacity and reduce undesired posttranslational modification.

Fig. 1 shows establishment BXP10-KEX2-PDI1-ERO1, the bacterial strain of a kind of process LAN KEX2, PDI1 and ERO1.By containing the KEX2-KanMX being operably connected to PGK1 promotor, be operably connected to the PDI-HphMX of PGK1 promotor, or the sequential NTS2-2 site (rDNA repeat in nontranscribed spacer) being integrated into BXP10 of the expression cassette of ERO-BsdMX being operably connected to PGK1 promotor, thus create following bacterial strain: BXP10-KEX2 (bacterial strain of process LAN KEX2), BXP10-KEX2-PDI1 (bacterial strain of process LAN KEX2 and PDI1) and BXP10-KEX2-PDI1-ERO1 (bacterial strain of process LAN KEX2, PDI1 and ERO1).

In order to build the expression cassette of KEX2, to be increased respectively KEX2ORF, PGK1 gene promoter (P from BXP10 genomic dna by PCR _pGK1) and ADH1 genetic transcription terminator sequence (T _aDH1), then use another PCR to react (" stitching " or " fusion " PCR reacts) and assemble.By the P of assembling _pGK1-KEX2-T _aDH1fragment is cloned in pRS314KanMX to generate pRS314KanMXpPGK1-KEX2.This plasmid in the end one is taken turns in PCR and is used as template to add and 5 ' of NTS2-2 integration site homology and 3 ' flanking sequence (being respectively 105bp and 101bp).By gained DNA fragmentation by being plated on the flat board containing G418 in Electroporation Transformation to BXP10 host strain.Confirm that G418 resistance clone is positive for site-specific integration further by bacterium colony PCR.

Build the plasmid pRS314HphMXpPGK1-PDI1 carrying the expression cassette of PDI1, it is by replacing the PDI1ORF of KEX2ORF and the KanMX district pcr amplification in pRS314KanMXpPGK1-KEX2 and Hygromycin B resistant mark HphMX.This plasmid in the end one is taken turns in PCR and is used as template to add and 5 ' of NTS2-2 integration site homology and 3 ' flanking sequence (being respectively 105bp and 101bp).By gained DNA fragmentation by being plated on the flat board containing hygromycin B in Electroporation Transformation to BXP10-KEX2 host strain.It is positive for confirming that Hygromycin B resistant is cloned for site-specific integration further by bacterium colony PCR.

By the genomic DNA amplification ERO1ORF of PCR from BXP10, be then cloned into further in pRS314pPGK1BsdMX to prepare pRS314BsdMXpPGK1-ERO1.This plasmid in the end one is taken turns in PCR and is used as template to add and 5 ' of NTS2-2 integration site homology and 3 ' flanking sequence (being respectively 105bp and 101bp).By gained DNA fragmentation by being plated on the flat board containing blasticidin S in Electroporation Transformation to BXP10-KEX2-PDI1 host strain.It is positive for confirming that blasticidin resistance is cloned for site-specific integration further by bacterium colony PCR.

Fig. 2 shows the NTS2-2 site that confirmation KEX2 and PDI1 is incorporated into BXP10, thus creates the Southern engram analysis of BXP10-KEX2 and BXP10-KEX2-PDI1 bacterial strain.Endogenous KEX2 is corresponded to for KEX2,2.6kb band copy, and 1.6kb band corresponds to the copy of Successful integration.Endogenous PDI1 is corresponded to for PDI1,1.3kb band copy, and 1.7kb band corresponds to the copy of Successful integration.

Fig. 3 shows the western blot analysis of PDI1 and KEX2, the process LAN of its display PDI1 and KEX2 in BXP10-KEX2-PDI1 clone.In 5 BXP10-KEX2-PDI1 clone, clone #2 shows the most high expression level of PDI1 and KEX2, is therefore selected as host strain to build BXP10-KEX2-PDI1-ERO1.

Then, by the pCID3610 Plastid transformation of host strain containing recombination fusion protein (" pCID3610 albumen "), this fusion rotein is made up of two human glucagon-like-peptides 1 (GLP-1, fragment 7-36 (A8G)) copied and recombinant human white protein (rHA).Each GLP-1 sequence, through modifying, wherein uses the naturally occurring L-Ala of glycine the position of substitution 8 to give the resistance to proteolysis.2nd GLP-1 sequence serves as the peptide spacer between a GLP-1 sequence and rHA.The non-glycosylated protein that pCID3610 albumen is made up of 645 amino acid and there is the molecular weight of 72,970.4Da.PCID3610 is described in detail in U.S. Patent No. 7,569,384, and it is complete is incorporated to herein.

Use the expression vector based on pSAC35, build pCID3610 plasmid at the HumanGenomeSciences of Rockville, MD.PSAC35 contains the LEU2 gene of yeast saccharomyces cerevisiae as selection marker thing, and it compensate for the leucine auxotrophy in BXP10.PSAC35 also clones and propagation with permission containing strong Yeast promoter (PRB1), unique cloning site (NotI) and the sequence from escherichia coli plasmid pUC9 in intestinal bacteria.In addition, pSAC35 is fissility (disintegrative) carrier, once it transforms in yeast, the sequence being derived from pUC9 is just cut by Site-specific recombinase.By FLP, this excision identifies that target thing (FLPrecognitiontarget, FRT) and yeast FLP (" flip ") recombinase realize from the expression of 2 micron plasmid.Other sections in pSAC35 comprise REP1 and the REP2 district of D-gene.REP1 and REP2 genes encoding helps the product of regulation and control plasmid copy number, and also works in plasmid is separated during cell fission.The product of D-gene increases FLP expression by alleviating the suppression caused by REP1 and REP2.PCID3610 is described in detail in U.S. Patent No. 7,569,384, and it is complete is incorporated to herein.

In the laboratory of Regius professor Dr.F.E.Baralle, be separated the full-length cDNA of HSA (HA) from human cDNA library and be cloned into plasmid pAT153ALB.Afterwards, pAT153ALB is modified so that be cloned in pSAC35 by introducing new restriction site by Delta.

By introducing the GLP-1-rHSA fusion gene of following assembling from pSAC35 construction of expression vector plasmid pCID3610.First, prepare the synthetic gene of encoding leader peptide and ripe GLP-1 variant, this variant has single A to G in the position 2 of mature peptide and replaces.Use the optimal codon reverse translation variant GLP-1 peptide for yeast, and use overlapping oligonucleotide to synthesize tandem copy via PCR.The gene of this synthesis is taken turns in PCR second and is used as template to add 5 ' and 3 ' restriction site, thus allows it to be cloned in 5 ' end of rHSA gene.Finally, signal coding sequence is connected to 5 ' end of GLP-1 construct.Gained fragment be connected to NotI site unique in pSAC35 and be transformed in DH5 α, obtaining expression vector pCID3610.Confirm the nucleotide sequence of pCID3610.Then, pCID3610 is transformed into again in DH5 α and is used for further expanding and isolated plasmid dna.

In order to the BXP10-KEX2-PDI1-ERO1 bacterial strain of construction expression pCID3610, by pCID3610 by Electroporation Transformation in BXP10-KEX2-PDI-ERO1, then by plating cells in ESFM2 agarose plate, select Leu+ bacterium colony afterwards 30C incubation plate 4 days.12 of transformant (12) bacterium colonies are rule to obtain single clone further on ESFM2 agarose plate.24 deep hole culture plate screenings are used by being used in ESFM2 substratum from a colony inoculation of often ruling.

Under agitation after 3 days incubations, SDS-PAGE to analyze in 12 cloned cultures the supernatant liquor of each.Fig. 4 shows the SDS-PAGE of 12 parts of supernatant samples.Then, from 12 clones, 4 clones (clone #2, clone #8, clone #10 and clone #12) are selected for test of fermenting further (clone of selection uses arrows in the diagram).Because the substratum from each hole is different in the evaporation of culture plate, 4 clones are selected in the consideration based on the band intensity on the final volume of every part of culture at the end of growth, the OD measurement of cell culture and SDS-PAGE gel.

Then, use fermenting procedure in the mini bio-reactor of DASGIP, run 4 clones selected, and by gained titre productive rate and protein quality and the BXP10 of expression pCID3610 those compared with, BXP10 is the host strain of a kind of only expressing K EX2, PDI1 and ERO1.The titre productive rate of the protein that Fig. 5 display produces in running fermentation and the analysis of protein quality.Protein quality is measured by the per-cent of following protein, and this protein has 6 extra amino acid (6-AA) due to the leader sequence cutting of poor efficiency at N-terminal.The remarkable increase (data expressing the BXP10 of pCID3610 do not show in Figure 5) in pCID3610 protein concn is presented at compared to the BXP10 only generating the expression pCID3610 reaching 1.6g/LpCID3610 albumen under identical fermentation condition, clone #2 and clone #8.In addition, compared to the 6-AA level in the BXP10 of the expression pCID3610 albumen that the protein of 4-7% has extra 6-AA, the level of 6-AA in all clones (protein of <1% has extra 6-AA) significantly reduces (data expressing the BXP10 of pCID3610 do not show in Figure 5).These results show that the process LAN of KEX2, PDI1 and ERO1 significantly improves host strain (BXP10) to produce the pCID3610 albumen more with more good quality.

Although the titre productive rate in clone #2 and clone #8 and 6-AA level are comparable, clone #8 is elected to be leading clone, because it shows slightly good result in fermentation runs.In order to confirm titre productive rate and the protein quality of the improvement of pCID3610, in 15L fermentation container, run clone #8.The average titer productive rate run batch from four (4) is 2.5g/L, and it is from the titre gain in yield 40-50% using the BXP10 expressing pCID3610.

Then two points of other freezing reserves that BXP10-KEX2-PDI1-ERO1 clones #8 are prepared.First freezing reserve (" ResearchCellBankVial ") is prepared by cultivating clone #8 in the 200mlESFM2 substratum containing all 3 kinds of microbiotic (G418, Totomycin and blasticidin).When cell culture reaches OD ₆₀₀when ~ 3.0, results, cleaning, re-suspended cell halving sampling are to prepare the freezing reserve of 20% trehalose.

Then, the cell from research cell bank bottle (ResearchCellBankVial) melted and cultivate in ESFM2 substratum at 30 DEG C and 250rpm.By measuring the OD of culture ₆₀₀monitoring culture density.The growth curve of Fig. 6 showed cell.At about OD ₆₀₀when=2.54, results, cleaning and re-suspended cell are to prepare the second freezing reserve (" Pre-MasterCellBank ").Fig. 7 display is from the growth curve of the cell of preparation master cell bank (Pre-MasterCellBank).

Then carry out by cell in vitro age and 15L PRODUCTION TRAITS the stability that test b XP10-KEX2-PDI1-ERO1 clones #8.In brief, gone down to posterity by 7 continuous print shaking flask steps by the clone #8 cell from preparation master cell bank, it corresponds to about 51 cell generation.Then, to seed cells in 15L fermentation container and to run via fermenting procedure.This fermenting process increases by 14 generations again.Supernatant liquor titre productive rate reaches 5.3g/L, and 6-AA level is lower than 1.5%.Data instruction BXP10-KEX2-PDI1-ERO1 clones #8 stable generation pCID3610 albumen after about 65 generations.

In order to build the expression cassette of KEX2, by PCR from BXP10 genomic dna each amplification PGK1 gene promoter (P respectively _pGK1), KEX2ORF and ADH1 genetic transcription terminator sequence (T _aDH1), then use another PCR to react (" stitching " or " fusion " PCR reacts) and assemble.By the P of assembling _pGK1-KEX2-T _aDH1fragment is cloned in pRS314KanMX to generate pRS314KanMXpPGK1-KEX2.Then, this plasmid in the end one is taken turns in PCR and is used as template to add and 5 ' of NTS2-2 integration site homology and 3 ' flanking sequence (being respectively 105bp and 101bp).By gained PCR fragment by being plated on the flat board containing G418 in Electroporation Transformation to BXP10 host strain.Confirm that G418 resistance clone is positive for site-specific integration further by bacterium colony PCR.

From BXP10 genomic DNA amplification PDI1ORF.This DNA fragmentation and Hygromycin B resistant mark HphMX are used for KEX2ORF and the KanMX district replaced in pRS314KanMXpPGK1-KEX2, it produces plasmid pRS314HphMXpPGK1-PDI1.Then, this plasmid in the end one is taken turns in PCR and is used as template to add and 5 ' of NTS2-2 integration site homology and 3 ' flanking sequence (being respectively 105bp and 101bp).By gained DNA fragmentation by being plated on the flat board containing hygromycin B in Electroporation Transformation to BXP10-KEX2 host strain.It is positive for confirming that Hygromycin B resistant is cloned for site-specific integration further by bacterium colony PCR.

Similarly, by the genomic DNA amplification ERO1ORF of PCR from BXP10, be then cloned into further in pRS314pPGK1BsdMX to prepare pRS314BsdMXpPGK1-ERO1.This plasmid in the end one is taken turns in PCR and is used as template to add and 5 ' of NTS2-2 integration site homology and 3 ' flanking sequence (being respectively 105bp and 101bp).By gained DNA fragmentation by being plated on the flat board containing blasticidin S in Electroporation Transformation to BXP10-KEX2-PDI1 host strain.It is positive for confirming that blasticidin resistance is cloned for site-specific integration further by bacterium colony PCR.

Claims (40)

1. a host cell, it comprises at least one coding and kills and wounds the heterologous nucleic acid sequence of expressing protein enzyme (Kex2p) or its fragment and/or variant and the heterologous nucleic acid sequence of at least one proteins encoded disulphide isomerase (Pdi1) or its fragment and/or variant.

2. the host cell of claim 1, it also comprises the heterologous nucleic acid sequence of at least one coding endoplasmic reticulum redox element (Ero1) or its fragment and/or variant.

3. the host cell of claim 1 or 2, wherein when cultivating described host cell in culture, the at least one gene product of at least one heterologous nucleic acid sequence described in described host cell expression or process LAN, described heterologous nucleic acid sequence coding is selected from following protein or its fragment and/or variant: Kex2p, Pdi1 and Ero1.

4. the host cell any one of claims 1 to 3, wherein said host cell is fungal cell.

5. the host cell of claim 4, wherein said fungal cell is yeast cell.

6. the host cell of claim 5, the genus of wherein said yeast cell is selected from lower group: yeast belong (Saccharomyces), genus kluyveromyces (Kluyveromyces), mycocandida (Candida), Pichia (Pichia), Schizosaccharomyces (Schizosaccharomyces), Hansenula (Hansenula), Ke Leke yeast (Kloeckera), perhaps prosperous yeast belong (Schwanniomyces) and Ye Shi yeast (Yarrowia).

7. the host cell any one of claim 1 to 6, wherein said yeast cell is yeast saccharomyces cerevisiae (S.cerevisiae).

8. the host cell any one of claims 1 to 3, wherein said host cell is mammalian cell.

9. the host cell any one of claim 1 to 8, the heterologous nucleic acid sequence of wherein said at least one encoded K ex2p or its fragment and/or variant is operably connected to the promotor that at least one is selected from lower group: TEF1, PRB1ADH1, ADH2, PYK1, PGK1, ENO, GAL1.10.7, GALS, MET25, CUP1, PHO5, tetO-CYC1, CaMV, HXT6, HXT7 and ARE.

10. the host cell any one of claim 1 to 8, the heterologous nucleic acid sequence of wherein said at least one coding Pdi1 is operably connected to the promotor that at least one is selected from lower group: TEF1, PRB1ADH1, ADH2, PYK1, PGK1, ENO, GAL1.10.7, GALS, MET25, CUP1, PHO5, tetO-CYC1, CaMV, HXT6, HXT7 and ARE.

Host cell any one of 11. claims 2 to 8, the heterologous nucleic acid sequence of wherein said at least one coding Ero1 is operably connected to the promotor that at least one is selected from lower group: TEF1, PRB1ADH1, ADH2, PYK1, PGK1, ENO, GAL1.10.7, GALS, MET25, CUP1, PHO5, tetO-CYC1, CaMV, HXT6, HXT7 and ARE.

Host cell any one of 12. claims 1 to 11, wherein said host cell also comprises the following genetic modification of at least one: pep4 proteolytic enzyme knocks out, ubc4 and/or ubc5 lower compared to wild-type host cells active, yps1 knocks out, hsp150 knocks out and knocks out with pmt1.

Host cell any one of 13. claims 1 to 12, it comprises the nucleic acid of at least one encoding recombinant polypeptide.

The host cell of 14. claims 13, the nucleic acid of wherein said at least one encoding recombinant polypeptide is included in plasmid.

The host cell of 15. claims 13, the nucleic acid integration of wherein said at least one encoding recombinant polypeptide is in the genome of described host cell.

Host cell any one of 16. claims 13 to 15, wherein said recombinant polypeptide has at least one disulfide linkage.

Host cell any one of 17. claims 13 to 16, wherein said recombinant polypeptide is albumin fusion proteins.

Host cell any one of 18. claims 13 to 17, wherein said recombinant polypeptide comprises at least one genetic fusion in the albuminised therapeutical peptide with GLP-1 activity.

Host cell any one of 19. claims 13 to 18, wherein said recombinant polypeptide has the polypeptide of the sequence iden with the aminoacid sequence shown in SEQIDNO:1 with at least 95%.

Host cell any one of 20. claims 13 to 19, wherein said recombinant polypeptide comprises the aminoacid sequence shown in SEQIDNO:1.

Host cell any one of 21. claims 13 to 20, wherein said recombinant polypeptide comprises leader sequence.

The host cell of 22. claims 21, wherein said leader sequence is KEX2 leader sequence or modified KEX2 leader sequence.

Host cell any one of 23. claims 13 to 22, wherein said recombinant protein comprises the modified leader sequence as shown in SEQIDNO:10.

24. 1 kinds of methods producing recombinant polypeptide, comprise the host cell cultivated any one of claim 13 to 23.

The method of 25. claims 24, it also comprises and reclaims described recombinant polypeptide from described substratum.

26. recombinant polypeptides prepared by the method for claim 24 or 25.

The recombinant polypeptide of 27. claims 26, wherein said recombinant polypeptide comprises the aminoacid sequence with SEQIDNO:1 with 99% sequence iden.

The recombinant polypeptide of 28. claims 27, wherein said recombinant polypeptide is made up of the aminoacid sequence of SEQIDNO:1.

29. pharmaceutical compositions, it comprises the recombinant polypeptide of claim 27 or 28.

The method of the patient that 30. 1 kinds of treatments have this to need, comprises the pharmaceutical composition of the claim 29 of administering therapeutic significant quantity.

The method of 31. claims 30, wherein said patient suffers from and is selected from following disease or situation: type i diabetes, type ii diabetes, glucose intolerance, hyperglycemia, Alzheimer's, obesity, cardiovascular disorder, congestive heart failure and retinopathy.

32. 1 kinds of host cells, it comprises at least one coding and kills and wounds the heterologous nucleic acid sequence of expressing protein enzyme (Kex2p) or its function fragment and/or variant, at least one proteins encoded disulphide isomerase (Pdi1) or the heterologous nucleic acid sequence of its function fragment and/or variant and the heterologous nucleic acid sequence of at least one coding endoplasmic reticulum redox element (Ero1) or its function fragment and/or variant.

The host cell of 33. claims 32, wherein said Kex2p or its function fragment and/or variant are the functional gene products of SEQIDNO:3.

The host cell of 34. claims 32, wherein said Pdi1 or its function fragment and/or variant are the functional gene products of SEQIDNO:5.

The host cell of 35. claims 32, wherein said Ero1 or its function fragment and/or variant are the functional gene products of SEQIDNO:7.

Host cell any one of 36. claims 32 to 35, wherein said host cell is yeast saccharomyces cerevisiae.

The host cell of 37. claims 32 or 36, it also comprises the nucleic acid of at least one encoding recombinant polypeptide.

The host cell of 38. claims 37, wherein said recombinant polypeptide comprises the polypeptide with the aminoacid sequence shown in SEQIDNO:1 with at least 99% sequence iden.

The host cell of 39. claims 37 or 38, wherein belong to same species with at least one and there is identical genetic modification but do not comprise the heterologous nucleic acid sequence that coding kills and wounds expressing protein enzyme (Kex2p) or its function fragment and/or variant, the host cell of the heterologous nucleic acid sequence of the heterologous nucleic acid sequence of at least one proteins encoded disulphide isomerase (Pdi1) or its function fragment and/or variant and at least one coding endoplasmic reticulum redox element (Ero1) or its function fragment and/or variant is compared, described host cell improves the titre productive rate of described recombinant polypeptide when cultivating in culture.

40. 1 kinds of host cells, when cultivating described host cell in culture, it is selected from the protein of Kex2p, Pdi1 and Ero1 or its fragment and/or variant compared to wild-type host cells process LAN at least two kinds, wherein said wild-type host cells belongs to same species and cultivates under same culture conditions, but process LAN at least two kinds is not selected from the gene product of Kex2p, Pdi1 and Ero1.