CN102533839A

CN102533839A - Method for performing fusion expression on protein containing disulfide bond

Info

Publication number: CN102533839A
Application number: CN2010106010812A
Authority: CN
Inventors: 张洁; 王建权; 余彩霞
Original assignee: SHANGHAI KAIYANG BIOTECHNOLOGY CO Ltd
Current assignee: SHANGHAI KAIYANG BIOTECHNOLOGY CO Ltd
Priority date: 2010-12-21
Filing date: 2010-12-21
Publication date: 2012-07-04
Anticipated expiration: 2030-12-21
Also published as: CN102533839B

Abstract

The invention relates to a method for performing fusion expression on a protein containing a disulfide bond, in particular to a method for performing fusion expression on a recombinant protein, in particular a eukaryotic recombinant protein of which the activity is closely related with the forming of the (intrachain and interchain) disulfide bond in an eukaryotic host cell. An exogenous recombinant protein is expressed in a pichia pastoris expression system by a method for fusing human protein disulphide isomerase (hPDI) or a mutant of the hPDI. The method is suitable for expressing a eukaryotic exogenous protein of which protein activity or protein folding (comprising forms of a monomer, a dimmer or a polymer) are resistant to the disulphide bond. The protein expressed by the mode can be secreted out of a cell easily, and is high in yield, products cannot be gathered on a large scale and can be captured and purified easily, and the obtained target protein is uniform in N tail end, and high in activity. The recombinant protein expressed by the method can be industrially produced on a large scale.

Description

Amalgamation and expression contains the method for disulfide bond protein matter

Technical field

The invention belongs to technical field of bioengineering, be specifically related to a kind of in eukaryotic host cell the methods and applications of correct express recombinant protein matter, particularly correctly express the closely-related eucaryon recombinant protein of formation of biological activity and disulfide linkage (the interior and interchain of chain).

Background technology

As far back as the seventies and eighties bibliographical information is arranged, find a kind of back translation enzyme (Post-Translational Enzymes)---Prolyl 4-hydroxylase, promptly prolyl-4-hydroxylase is called for short P4H.It can be to the characteristic Tumor-necrosis factor glycoproteins (Gly-X-Y) of collagen family protein _nIn Y carry out hydroxylation and modify (when Y is proline residue), become oxyproline.Have only proline residue to be modified to after 4 oxyprolines when some amount; Three polypeptied chains of collagen protein could correctly be folded into the triple-helix structure territory; That is to say and have only by hydroxylated collagen protein just solvablely, could correctly be folded into activated albumen.(Prockop?et?al.，N.Engl.J.Med.311：376-386(1984).)

Prolyl-4-hydroxylase, P4H is with α ₂β ₂Tetrameric form exists.(Berg et al., J.Biol.Chem.248:1175-1192 (1973); Tuderman e t al., Eur.J.Biochem.52:9-16 (1975) .) wherein, the α subunit includes the avtive spot of 4 prolyl hydroxylations, is the part with catalytic activity.But the α subunit is soluble when not having the β subunit to exist, and does not also have activity; And the β subunit separately can the catalysis disulfide linkage formation, improve proteic stability, help proteic correct foldingly, be known as disulfide bond isomerase (protein disulfide isomerase, i.e. PDI).After forming the tetramer, the β subunit still remains with 50% disulfide linkage heterogeneous activity.(Pihlajaniemi?et?al.，Embo?J.6：643-649(1987)；Parkkonen?et?al.，Biochem.J.256：1005-1011(1988)；Koivu?et?al.，J.Biol.Chem.262：6447-6449(1987))。

Because the collagen family protein is had the posttranslational modification effect, in recent years, prolyl-4-hydroxylase becomes the focus of countries in the world research.All there is correlative study in states such as the U.S., Canada, Finland, Australia, Japan.Wherein, the scientist Kari.Kivirikko of Finland is having very distinguished achievement in research aspect the relation of prolyl-4-hydroxylase and all types of collagen proteins, and he has many pieces of papers and related patent U.S. Patent No. (U.S.) to deliver.

Kari.Kivirikko is published in July 28 (U.S.Pat.NO.2005/0164345A1) in 2005 about nearest one piece of patent (U.S.) of prolyl-4-hydroxylase.In this patent, he has described in yeast saccharomyces cerevisiae expression system, methanol yeast expression system, insect cell expression system, assists the method for express recombinant human collagen or procollagen with recombinant human prolyl-4-hydroxylase (P4H).Wherein right: recombinant human collagen albumen Type I, TypeII, TypeIII, TypeIV, TypeXIII, TypeX V, TypeX VIII express in the expressed in insect cells system and recombinant human collagen albumen TypeIII expresses the detailed introduction of contrasting in yeast expression system.

Another piece is published in USP (U.S.Pat.NO.6 in 2002; 451; 557B1), be example then through express recombinant human collagen TypeIII, described in detail with yeast host bacterium co expression recombinant human prolyl-4-hydroxylase (P4H) and proteic method of recombinant human collagen.

And one piece of nearest USP that is published in this year (2010); Be by Taiwan's scholars Chou; (U.S.Pat.NO.7279329) of et al. makes; Describe utilization and the recombinant human prolyl-coexpression of 4-hydroxylase (P4H) in insect cell line, come the method in the NC1 district (C-terminal noncollagenous (NC1) domain) of secreting, expressing XXI collagen type.

More than these work, pointed out recombinant human prolyl-4-hydroxylase (P4H) in bacterium, yeast and insect cell expression system, the promoter action during to the protein expression of collagen protein family.Also for we express the collagen proteinoid, and some other closely-related eucaryon recombinant protein of formation active and disulfide linkage, a thinking is provided.

But we find in actual experiment work, make up the co-expression carrier of recombinant human prolyl-two subunits of 4-hydroxylase (P4H) (α and β), and method is comparatively loaded down with trivial details; And; In industry, use in the more eukaryotic expression system pichia spp host bacterium (Pichia pastoris); The independent expression amount of recombinant human prolyl-4-hydroxylase (P4H) is very low, that tetramer albumen is difficult to is to be detected (Myllyharju et al., EMBO is J.16; 1173-1180,1997).Realize that tetramer albumen and the co expression of target protein in same host cell also are difficult to the effect that reaches satisfied.In order to address this problem, we have turned to β subunit wherein to research emphasis by people's prolyl-4-hydroxylase (P4H) tetramer albumen, just recombinant human protein's matter disulfide bond isomerase (human protein disulphide isomerase).

For many albumen, the formation of disulfide linkage is correctly folding basis (Derman et al. (1993) Science262:1744 of albumen normally.7)。And the correctly folding process need of albumen relies on the booster action of other protein factor or folding enzymes, and this proteinoid factor or folding enzymes are known as Chaperones Molecular (foldase and chaperone).Protein disulfide isomerase (protein disulfide isomerase; PDI) be a kind of in the folding enzymes; Its family member extensively is present in the Mammals; They are common in endocytoplasmic reticulum, and main function is formation, isomery and the reduction of the disulfide linkage of new polypeptide chain in the catalysis endoplasmic reticulum, and then help peptide chain correctly to be folded into active tertiary structure.It is to find to help the highest folding enzymes of protein folding activity at present.In addition at aspects such as Endoplasmic Reticulum Associated Protein Degradation approach ERAD protein transport, calcium homeostasis, angtigen presentation and poisoning intrusions they also play an important role (ELLGAARDL, RUDDOCK L W.EMBO Rep, 2005,6:28-32.).Based on PDI albumen as prolyl-proteic β subunit of 4-hydroxylase (P4H) tetramer; With these key characteristics as folding enzymes; We believe; It should be able to be from engineered aspect, for we provide one to solve the biotechnology research and development and produce inclusion body (Inclusion Bodies, IB) method of problem that extensively exists in the process of protein medicaments.

Pichia spp (Pichia pastoris) is that the more eukaryotic expression system of use is gone up in industry, owing to characteristics such as its cultivation are convenient, and output is high, and cost is low, is used for doing the eucaryon host of expression foreign protein widely.Though pichia spp itself has PDI albumen (TIAN G, XIANG S equally; NOIVA R, et al.Cell, 2006; 124:61-73.), can auxiliary expression proteic folding, but in practical application, find; Still old a large amount of foreign proteins are not expressed in pichia spp, and perhaps expression amount is extremely low.Adopt multiple copied or use method such as yeast preference codon instead and still can not change this situation.

After repeatedly attempting, we try to seek reason from recombinant protein itself.Because our recombinant protein derives from Mammals mostly; For example the albumen in people source is just very extensive; And extensively existing oneself protein disulfide isomerase in the mammalian cell, these enzymes and saccharomycetic protein disulfide isomerase homology are not high.Infer that thus the recombinant protein in that the pichia spp system is expressed under the situation of the folding enzymes that lacks corresponding source, can't be folded into their natural structure, thereby chat problem on having caused.So we imagine; In yeast host; Use the mode express recombinant people source protein matter disulfide bond isomerase (hPDI) and the target protein (particularly people's source protein) of fusion, be about to human protein disulfide bond isomerase full-length proteins or its two mutants, directly perhaps to pass through connection peptides Linker ways of connecting; Be connected the N end of target protein, realize amalgamation and expression.The benefit that designs like this, the one, can utilize the high expression level characteristic of hPDI albumen in pichia spp, see my patent (application number: 201010567613.5), as homing sequence, take hPDI albumen out of the target protein secreting, expressing for details; Two, the hPDI albumen after the sudden change has-the HDEL tail, more helps locating the endoplasmic reticulum of yeast host cell, makes target protein form correct disulfide linkage down, and then be folded into correct conformation the auxiliary of hPDI; Moreover, merge at the hPDI of target protein N-terminal albumen, also avoided the N end heterogeneity phenomenon (Boehm, T., et al.Yeast 15,563.572) of Pichia anomala expression product, protected the integrity of target protein.

This imagination to the amalgamation and expression that several albumen Endostatin, Tumstatin, Canstatin, Arrestin, the Angiostatin that comes from people's collagen family carries out said mode, has obtained checking through us one by one.

Summary of the invention

Content of the present invention provides a kind of method, particularly active structure of external source recombinant protein and closely-related eucaryon recombinant protein of formation of disulfide linkage (the interior and interchain of chain) of in eukaryotic host cell, expressing.

Method of the present invention comprises:

Preparation can express, the hPDI full-length gene of biologically active;

2. sudden change hPDI full-length gene, and be connected with connection peptides, hPDI obtained ₄₉₁-L mutator gene;

Preparation can express, people's endostatin (Endostatin) full-length gene of biologically active;

4. with hPDI ₄₉₁-L mutator gene and people's endostatin (Endostatin) full-length gene and expression vector reorganization;

5. with said carrier transfection host cell;

6. cultivate said host cell;

7. purified fusion protein, and after enzyme is cut, be further purified target protein.

The present invention clones hPDI cDNA full-length gene with the method for PCR from people's liver cDNA library.Said hPDI cDNA full-length gene corresponding amino acid sequence is shown in sequence table SEQ-1.

HPDI cDNA full-length gene of the present invention source is not limited to any specific human body cell, cell strain or tissue, as long as it contains hPDI mRNA.

The two mutants of recombinant human protein's matter disulfide bond isomerase of the present invention is not limited to the two mutants of which kind of form of this enzyme, as long as it helps the expression of target protein in eucaryon host with correct folding.In a preferred embodiment, the two mutants that the present invention uses has removed the signal sequence (1-17aa) of recombinant human protein's matter disulfide bond isomerase self, and proteic end is changed into-HDEL by-KDEL, and the aminoacid sequence after the sudden change is shown in sequence table SEQ-2.

Expression product of the present invention is secreted into outside the born of the same parents, is present in the host cell nutrient solution supernatant.Centrifugal removal host cell can separate and purified fusion protein from the nutrient solution supernatant.Fusion rotein can separate the target protein that obtains purifying after cutting through the enteropeptidase enzyme.Said expression product recombinant human endostatin (Endostatin) protein amino acid sequence is shown in sequence table SEQ-4.The present invention carries out mass production with the mode of amalgamation and expression to the albumen recombinant human endostatin (Endostatin) of collagen protein family, and the protein product of gained is secretion, soluble protein.Through after the simple purification, can obtain soluble, terminal homogeneous, active very high target protein.This method is compared with known method, and not only protein-active is higher, and output is higher, and the albumen homogeneous, and safety is more suitable for being used for scale operation.

Method of the present invention is not limited to any specific albumen, and needing only it can merge with recombinant human protein's matter disulfide bond isomerase, and carries out correct folding down assisting of recombinant human protein's matter disulfide bond isomerase.

The intestinal bacteria that this patent is established and arrived have been submitted preservation on December 16th, 2010.Depositary institution's title: Chinese typical culture collection center; Preservation address: China, Wuhan City, Hubei Province, Wuhan University; Culture presevation number is CCTCC M 2010353; Classification called after: ETEC K-12/pPICZalpha-hPDI (491)-Endostatin E2; Escherichia coliK-12/pPICZalpha-hPDI (491)-Endostatin E2.

The molecular biology operation is all with reference to " molecular cloning experiment guide " (third edition, Science Press, 2002) in the above-mentioned technological method of the present invention.

Description of drawings

Fig. 1:

The gene constructed process synoptic diagram of fusion expression plasmid ppICZa-hPDI-L-Endostatin.

Fig. 2:

2-1: pcr amplification endostatin gene from people liver cDNA Lib, No. 2 sample amplification gone out size about 600bps band, conform to endostatin gene size.

2-2,2-3: the fusion expression plasmid ppICZa-hPDI-L-Endostatin enzyme of structure is cut evaluation: No. 3 samples, plasmid is with Xho I single endonuclease digestion.Because the hPDI gene has two Xho I sites, add that plasmid ppICZa carrier part has an Xho I site, so Xho I single endonuclease digestion plasmid can downcut the band of a 500bps and a 900bps, accord with expectation as a result; No. 4 samples, plasmid is with EcoR I+Not I double digestion.On the carrier that makes up, EcoR I is positioned on the Linker, and Not I is that the site is inserted in the downstream of gene, so double digestion can downcut the Endostatin gene, just the band about 600bps, accord with expectation as a result.Figure can confirm that the plasmid ppICZa-hPDI-L-Endostatin that makes up is correct from the angle that enzyme is cut thus.

Fig. 3:

The plasmid ppICZa-hPDI-L-Endostatin that makes up is with the sequencer address of 3 ' AOX primer, and sequencing result shows, the gene Endostatin sequence and the correct position of insertion.

Fig. 4:

The plasmid ppICZa-hPDI-L-Endostatin that makes up; After changing methanol yeast host bacterium X33 over to through electricity, PE1, PE2, three mono-clonal samples of PE8# have been filtered out, methanol induction 48 hours; With the inducing culture supernatant of three samples concentrate, desalination; And after EK enzyme enzyme cuts, walk concentration respectively and be 8% SDS-PAGE reduction and non-reduced electrophoresis, comparative result.

M: albumen marker

The 1:PE2# sample is induced preceding sample supernatant

The 2:PE1# sample is induced the back supernatant

After the 3:PE1# sample was induced, supernatant was cut 1 hour result through concentrating and desalinating with EK enzyme enzyme

The 4:PE2# sample is induced the back supernatant

After the 5:PE2# sample was induced, supernatant was cut 1 hour result through concentrating and desalinating with EK enzyme enzyme

The 6:PE8# sample is induced the back supernatant

After the 7:PE8# sample was induced, supernatant was cut 1 hour result through concentrating and desalinating with EK enzyme enzyme

(the 1-8 sample is the reduction electrophoresis result)

M: albumen marker

The 8:PE2# sample is induced preceding sample supernatant

The 9:PE2# sample is induced the back supernatant

After the 10:PE2# sample was induced, supernatant was cut 1 hour result through concentrating and desalinating with EK enzyme enzyme

The 11:PE8# sample is induced the back supernatant

After the 12:PE8# sample was induced, supernatant was cut 1 hour result through concentrating and desalinating with EK enzyme enzyme

(the 8-12 sample is non-reduced electrophoresis result)

Electrophoresis result shows; The PE2# sample of screening after 48 hours, has occurred near the protein band (hPDI-L-Endostatin albumen size be 78.3KDa) of the size 75KDa position in methanol induction the supernatant, after process EK enzyme enzyme is cut 1 hour; Protein band is cut into two sections; Wherein bigger one section is slightly larger than 52KDa (hPDI-L albumen size is 58.2KDa), and thus, we can think that the expressed proteins band is target protein hPDI-L-Endostatin.

Embodiment

Embodiment 1: design, construction recombination plasmid, expressed fusion protein

(1) clone hPDI ₅₀₈The cDNA gene

With reference to the hPDI gene order, the design primer is used PCR method, amplification total length hPDI gene from commercialization people liver cDNA library.Amplified production cuts out from restriction enzyme Xho I and NotI site, with Pichia anomala expression plasmid pPICZ α reorganization, construction expression plasmid pPICZ α-hPDI ₅₀₈, transform NovaBlue host bacterium.The extracting plasmid again through corresponding restriction endonuclease sites restriction analysis, with characteristic fragment screening plasmid, is used nucleotide sequence analysis then, confirms that the position and the sequence of recombination is correct, obtains to have the positive colony of hPDI full-length gene.

The restriction enzyme that the present invention adopted is from Takara company, and commercialization people liver cDNA library is from Biochain company, and Pichia pastoris strain X 33, pichia spp multiple copied expression vector pPICZ α are from Invitrogen company.

(2) sudden change total length hPDI gene

Use conventional PCR method, through synthetic upstream and downstream primer, the sudden change full-length gene.Being reflected on the amino acid levels is exactly to remove the proteic signal sequence of hPDI (1-17aa), and 3 ' end of mutator gene-KDEL becomes-HDEL, with reference to my patent (application number: 201010567613.5).And, use the mode of conventional PCR equally, the hPDI after sudden change with other synthetic primer ₄₉₁3 ' end of gene adds catenation sequence Linker (SEQ-3), promptly obtains hPDI ₄₉₁The PCR product of-L.

With amplified production and the reorganization of pGEM-T carrier, make up new expression plasmid pGEM-T-hPDI ₄₉₁-L transforms NovaBlue host bacterium.The extracting plasmid again through corresponding restriction endonuclease sites restriction analysis, with characteristic fragment screening plasmid, is used nucleotide sequence analysis then, confirms that the position and the sequence of recombination is correct, obtains to have hPDI ₄₉₁The positive colony of-L gene.

The pGEM-T carrier that the present invention adopted is from Promega company.

(3) human cloning endostatin (endostatin) gene

With reference to the online collagen protein family that announces several proteic gene order; The design primer; Use PCR method, endostatin (endostatin) gene of amplification collagen protein family from commercialization people liver cDNA people from library is with amplified production and the reorganization of pGEM-T carrier; Make up new expression plasmid pGEM-T-Endostatin, transform NovaBlue host bacterium.The extracting plasmid again through corresponding restriction endonuclease sites restriction analysis, with characteristic fragment screening plasmid, is used nucleotide sequence analysis then, confirms that the position and the sequence of recombination is correct, obtains to have the positive colony of Endostatin gene.

(4) make up plasmid, screening and expressed fusion protein hPDI ₄₉₁-L-Endostatin

With making up successful plasmid pGEM-T-hPDI ₄₉₁-L and pGEM-T-Endostatin use XhoI+BamHI, BamHI+NotI double digestion respectively, and enzyme is cut product and Pichia anomala expression plasmid pPICZ α reorganization, construction expression plasmid pPICZ α-hPDI ₄₉₁-L-Endostatin transforms NovaBlue host bacterium.The extracting plasmid again through corresponding restriction endonuclease sites restriction analysis, with characteristic fragment screening plasmid, is used nucleotide sequence analysis then, confirms that the position and the sequence of recombination is correct, obtains to have hPDI ₄₉₁The positive colony of-L-Endostatin fusion gene.

Plasmid pPICZ α-hPDI that extracting builds ₄₉₁-L-Endostatin, after restriction enzyme SacI linearizing, electroporation changes among the pichia spp wild-type host bacterium X33.Through the screening of Zeocin resistant panel, obtain positive colony.Positive colony is seeded to the BMGY substratum, wherein contains 1% yeast extract, 2% peptone, 100mM phosphate buffered saline buffer (pH6.0), 1.34%YNB, (4 * 10 ^-5) % vitamin H and 1% glycerine, 30 ℃ are cultured to thalline optical density(OD) OD _600nmReach at 2～6 o'clock, centrifugal removal nutrient solution is diluted to OD with bacterial sediment with the compound substratum BMMY of methyl alcohol _600nmTo 1, substratum BMMY contains 1% yeast extract, 2% peptone, 100mM phosphoric acid buffer (pH 6.0), 1.34%YNB, (4 * 10 ^-5) the % vitamin H, 0.5% methyl alcohol.30 ℃ of inducing culture 72 hours, the methyl alcohol of taking a sample and add was kept its concentration 0.5% in per 12 hours, kept the nutrient solution supernatant, did the non-reduced electrophoresis of 8%SDS-PAGE with supernatant, and after the Xylene Brilliant Cyanine G R-250 dyeing, purity, molecular weight are decided in scanning.The result shows that behind the methanol induction, supernatant has the band that concentrates in corresponding molecular weight position.Confirm roughly that through scanning target protein accounts for the supernatant thalline and expresses more than 50% of total protein.

Embodiment 2: the purifying of fusion rotein

1.CM Sepharose Fast Flow column chromatography

With 50mM HAc-NaAc (pH 4.2) balance pillar; Being diluted to electricity to the appearance liquid that contains target protein leads consistent with level pad; And regulate pH to 4.2, and begin to go up appearance, wash 6 column volumes with level pad behind the end of the sample; Use 50mMHAc-NaAc (pH4.0)+1M NaCl wash-out again, collect 2 column volumes.

2.Chelating Sepharose Fast Flow chromatography

1 times of CM elutriant dilute with water; Regulate pH to 7.4 with sodium hydroxide again; On used the good Chelating Sepharose Fast Flow post of 50mMTris-HCl (pH 7.4)+0.5M NaCl balance, with 6 column volumes of level pad flushing, use 50mMTris-HCl (pH 7.4)+20mM imidazoles flush away impurity again behind the end of the sample; Use 50mMTris-HCl (pH7.4)+200mM imidazoles wash-out at last, collect 2 column volumes.

3.Chelating Sepharose Fast Flow elutriant is with the ultrafiltration of 10KD ultra-filtration membrane.

4. albumen was cut 1 hour at 37 ℃ of enzymes with ox source enteropeptidase (rEK).

5.SP Sepharose Fast Flow column chromatography

With 50mMTris-HCl (pH 7.0) balance pillar; The electricity of cutting back solution to enzyme lead with pH regulator to consistent with level pad; Begin to go up appearance, with 6 column volumes of level pad flushing, use 50mMTris-HCl (pH 7.0)+0.3MNaCl to wash impurity again behind the end of the sample; Use 50mMTris-HCl (pH 7.0)+1MNaCl wash-out at last, collect 3 column volumes.

6.Sephadex obtain soluble target protein Endostatin after the G-25 desalination.

The protein molecule sequence

The explanation of SEQ-1 sequence:

(1) sequence description: human protein disulfide bond isomerase full-length proteins

(2) molecule type: protein

(3) sequence signature:

A. length: 508aa

B. molecular weight: 57.1KDa

C. iso-electric point: 4.76

1 MLRRALLCLA?VAALVRADAP?EEEDHVLVLR?KSNFAEALAA?HKYLLVEFYA

51?PWCGHCKALA?PEYAKAAGKL?KAEGSEIRLA?KVDATEESDL?AQQYGVRGYP

101TIKFFRNGDT?ASPKEYTAGR?EADDIVNWLK?KRTGPAATTL?PDGAAAESLV

151ESSEVAVIGF?FKDVESDSAK?QFLQAAEAID?DIPFGITSNS?DVFSKYQLDK

201DGVVLFKKFD?EGRNNFEGEV?TKENLLDFIK?HNQLPLVIEF?TEQTAPKIFG

251GEIKTHILLF?LPKSVSDYDG?KLSNFKTAAE?SFKGKILFIF?IDSDHTDNQR

301ILEFFGLKKE?ECPAVRLITL?EEEMTKYKPE?SEELTAERIT?EFCHRFLEGK

351IKPHLMSQEL?PEDWDKQPVK?VLVGKNFEDV?AFDEKKNVFV?EFYAPWCGHC

401KQLAPIWDKL?GETYKDHENI?VIAKMDSTAN?EVEAVKVHSF?PTLKFFPASA

451DRTVIDYNGE?RTLDGFKKFL?ESGGQDGAGD?DDDLEDLEEA?EEPDMEEDDD

501QKAVKDEL

SEQ-2

(4) sequence description: human protein disulfide bond isomerase two mutants

(5) molecule type: protein

(6) sequence signature:

A. length: 491aa

B. molecular weight: 55.3KDa

C. iso-electric point: 4.69

1?DAPEEEDHVL?VLRKSNFAEA?LAAHKYLLVE?FYAPWCGHCK?ALAPEYAKAA

51GKLKAEGSEI?RLAKVDATEE?SDLAQQYGVR?GYPTIKFFRN?GDTASPKEYT

101AGREADDIVN?WLKKRTGPAA?TTLPDGAAAE?SLVESSEVAV?IGFFKDVESD

151SAKQFLQAAE?AIDDIPFGIT?SNSDVFSKYQ?LDKDGVVLFK?KFDEGRNNFE

201GEVTKENLLD?FIKHNQLPLV?IEFTEQTAPK?IFGGEIKTHI?LLFLPKSVSD

251YDGKLSNFKT?AAESFKGKIL?FIFIDSDHTD?NQRILEFFGL?KKEECPAVRL

301ITLEEEMTKY?KPESEELTAE?RITEFCHRFL?EGKIKPHLMS?QELPEDWDKQ

351PVKVLVGKNF?EDVAFDEKKN?VFVEFYAPWC?GHCKQLAPIW?DKLGETYKDH

401ENIVIAKMDS?TANEVEAVKV?HSFPTLKFFP?ASADRTVIDY?NGERTLDGFK

451KFLESGGQDG?AGDDDDLEDL?EEAEEPDMEE?DDDQKAVHDE?L-

SEQ-3

(7) sequence description: connection peptides

(8) molecule type: polypeptide

(9) sequence signature:

A. length: 30aa

B. molecular weight: 2.8KDa

C. iso-electric point: 5.72

1GTEFGGGGSG?GGGSHHHHHH?GGGGSDDDDK

//

SEQ-4

(10) sequence description: human endostatin full-length proteins (Endostatin)

(11) molecule type: protein

(12) sequence signature:

A. length: 183aa

B. molecular weight: 20.1KDa

C. iso-electric point: 9.30

1 HSHRDFQPVL?HLVALNSPLS?GGMRGIRGAD?FQCFQQARAV?GLAGTFRAFL

51?SSRLQDLYSI?VRRADRAAVP?IVNLKDELLF?PSWEALFSGS?EGPLKPGARI

101FSFDGKDVLR?HPTWPQKSVW?HGSDPNGRRL?TESYCETWRT?EAPSATGQAS

151SLLGGRLLGQ?SAASCHHAYI?VLCIENSFMT?ASK

SEQ-5

(13) sequence description: fusion rotein (hPDI-L-Endostatin)

(14) molecule type: protein

(15) sequence signature:

A. length: 705aa

B. molecular weight: 78.3KDa

C. iso-electric point: 5.08

1 MDAPEEEDHV?LVLRKSNFAE?ALAAHKYLLV?EFYAPWCGHC?KALAPEYAKA

51?AGKLKAEGSE?IRLAKVDATE?ESDLAQQYGV?RGYPTIKFFR?NGDTASPKEY

101TAGREADDIV?NWLKKRTGPA?ATTLPDGAAA?ESLVESSEVA?VIGFFKDVES

151DSAKQFLQAA?EAIDDIPFGI?TSNSDVFSKY?QLDKDGVVLF?KKFDEGRNNF

201EGEVTKENLL?DFIKHNQLPL?VIEFTEQTAP?KIFGGEIKTH?ILLFLPKSVS

251DYDGKLSNFK?TAAESFKGKI?LFIFIDSDHT?DNQRILEFFG?LKKEECPAVR

301LITLEEEMTK?YKPESEELTA?ERITEFCHRF?LEGKIKPHLM?SQELPEDWDK

351QPVKVLVGKN?FEDVAFDEKK?NVFVEFYAPW?CGHCKQLAPI?WDKLGETYKD

401HENIVIAKMD?STANEVEAVK?VHSFPTLKFF?PASADRTVID?YNGERTLDGF

451KKFLESGGQD?GAGDDDDLED?LEEAEEPDME?EDDDQKAVHD?ELGTEFGGGG

501SGGGGSHHHH?HHGGGGSDDD?DKHSHRDFQP?VLHLVALNSP?LSGGMRGIRG

551ADFQCFQQAR?AVGLAGTFRA?FLSSRLQDLY?SIVRRADRAA?VPIVNLKDEL

601LFPSWEALFS?GSEGPLKPGA?RIFSFDGKDV?LRHPTWPQKS?VWHGSDPNGR

651RLTESYCETW?RTEAPSATGQ?ASSLLGGRLL?GQSAASCHHA?YIVLCIENSF

701MTASK

//

Claims

1. the method for an amalgamation and expression recombinant protein in eukaryotic host cell, the recombinant protein that it is characterized in that amalgamation and expression have a kind of among the structure:

a).P-X

b).P-L-X

c).MP-X

d).MP-L-X

Wherein, P represents recombinant human protein's matter disulfide bond isomerase (human protein disulphide isomerase);

MP represents recombinant human protein's matter disulfide bond isomerase two mutants (mutant of human protein disulphide isomerase);

L representes connection peptides (linker sequence);

X represents target protein (interest protein).

2. the method for an amalgamation and expression recombinant protein in eukaryotic host cell, the recombinant protein that it is characterized in that amalgamation and expression have a kind of among the structure:

a).P-X

b).P-L-X

c).MP-X

d).MP-L-X

Wherein, P represents recombinant human protein's matter disulfide bond isomerase (human protein disulphide isomerase); MP represents recombinant human protein's matter disulfide bond isomerase two mutants (mutant of human protein disulphide isomerase);

L representes connection peptides (linker sequence);

X represents target protein (interest protein).

3. method according to claim 1, the gene clone of the recombinant human protein's matter of wherein encoding disulfide bond isomerase (being P albumen) is from the total RNA of human tissue cell.

4. according to claim 2, the total RNA of human tissue cell wherein is not limited to the specific cell of human body, cell strain or tissue, as long as it contains hPDI mRNA.

5. said with 3 according to claim 2, the aminoacid sequence of recombinant human protein's matter disulfide bond isomerase wherein (being P albumen) is SEQ-1.

6. method according to claim 1, recombinant human protein's matter disulfide bond isomerase two mutants wherein, i.e. MP albumen is not limited to the two mutants of which kind of form of this enzyme, as long as it helps the expression of target protein in eucaryon host with correct folding.

7. according to claim 1 and 3 described methods; Recombinant human protein's matter disulfide bond isomerase two mutants (being MP albumen) wherein is 1-17 amino-acid residue amputation with the coded signal sequence of the human protein disulfide bond isomerase (hPDI) of total length; And Lys (505) sported His (505); Obtain one and contain 491 amino acid whose polypeptied chains, it is DAPEEEDH-that its N-holds preceding 8 amino-acid residues, and 6 amino-acid residues in C-end end are-and AVHDEL (is r-hPDI ₄₉₁), its aminoacid sequence is SEQ-2, culture presevation numbering: CCTCC M 2010318.

8. method according to claim 1, linker sequence (L) sequence wherein is not limited to any connection peptides sequence, and it can include (Gly ₄Ser) _n, (G1y) _n, (Ser) _n, (His) ₆, enteropeptidase (rEK) recognition sequence DDDDK ^↓, marmor erodens proteolytic enzyme (rTEV) recognition sequence ENLYFQ ^↓G, zymoplasm (Thrombin) recognition sequence LVPR ^↓The recognition sequence IEGR of GS or factor Xa ^↓Various combinations, connect as long as it helps albumen.

9. according to claim 1 and 7 described methods, connection peptides linker sequence (L) wherein has the aminoacid sequence shown in the SEQ-3.

10. method according to claim 1; Target protein wherein, promptly X protein is not limited to any specific albumen; As long as it can merge with recombinant human protein's matter disulfide bond isomerase, and carry out correct folding down assisting of recombinant human protein's matter disulfide bond isomerase.

11. according to claim 1 and 9 described methods, target protein wherein, i.e. X protein comprises Endostatin, Tumstatin, Arrestin, Canstatin and the Angiostatin of collagen protein family and their truncation type or mutein.

12. according to claim 1,9 and 10 said, target protein wherein, i.e. X protein, when it refered in particular to the Endostatin full-length proteins, the aminoacid sequence of its full-length proteins was SEQ-4; During amalgamation and expression, the aminoacid sequence of excretory fusion rotein is SEQ-5, culture presevation numbering: CCTCC M 2010353.

13. method according to claim 1, eukaryotic host cell wherein are not limited to any specific host cell, as long as it can express described fusion rotein.

14. according to claim 1 and 12 described methods, wherein said eukaryotic host cell is a Pichia pastoris strain X 33.