CN102533841A

CN102533841A - Method for increasing expression of bacillus thuringiensis(Bt) insecticidalcrystalprotein in hansenula polymorpha

Info

Publication number: CN102533841A
Application number: CN2012100302182A
Authority: CN
Inventors: 刘德虎; 王楠; 李刚强; 刘树鹏
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2012-02-13
Filing date: 2012-02-13
Publication date: 2012-07-04
Anticipated expiration: 2032-02-13
Also published as: CN102533841B

Abstract

The invention discloses a method for increasing expression of Bt insecticidalcrystalprotein in hansenula polymorpha and relates to the field of genetic engineering, in particular to a method for realizing constitutive high-efficiency expression of a Bt fusion protein gene in hansenula polymorpha cells through a composite enhancer, which comprises: 1) using one composite enhancer to increase the expression of the Bt fusion protein after codon improvement in hansenula polymorpha; 2) controlling the Bt fusion protein gene to express in the hansenula polymorpha in a constitutive manner by using a glyceraldehyde-3-phosphate dehydrogenase promoter in pichia pastoris; 3) providing optimized hansenula polymorpha fermentation, culture and growth conditions; and 4) quickly purifying the recombinant heterologous protein by using nanofiltration and nickel column affinity chromatography. The Bt recombinant fusion protein prepared by the method can be used as a candidate for protein standard substance in the fields of protein traceability study and the like.

Description

A kind of method that improves Bt insecticidal proteins expression amount in debaryomyces hansenii

Technical field

The present invention relates to the genetically engineered field; Be specifically related to use the method for a compound enhanser to realize that the Bt antigen-4 fusion protein gene efficiently expresses with the composing type mode in many types of debaryomyces hansenii cell, comprising: 1) use a compound enhanser to improve the output of Bt antigen-4 fusion protein gene in debaryomyces hansenii after codon optimized; 2) adopt pichia spp glyceraldehyde-3-phosphate dehydrogenase promoter regulation Bt antigen-4 fusion protein gene mode with composing type in debaryomyces hansenii to express; 3) the debaryomyces hansenii fermentation culture growth conditions after the optimization; 4) utilize purify the fast method of this recombinant exogenous protein of nanofiltration and nickel post affinity chromatography.And the Bt recombination fusion protein of method preparation can be used as protein reference material alternatives and is applied to the protein fields such as research of tracing to the source thus.

Background technology

Bt is the abbreviation of Tribactur (Bacillus thuringiensis), belongs to earth bacillus on the Gram-positive.The Premeabilisation of cells that this bacteriogenic Bt desinsection toxalbumin can be destroyed in the insect gut is pressed, and causes digestive disorders, finally causes insect death.The Bt gene is typical case's representative of first-generation anti insect gene, and its expressed proteins is referred to as the Cry crystallin.According to the difference of insecticidal spectrum and amino acid sequence homology, the Bt gene is divided into different types, and wherein Cry1Ab and Cry1Ac have specificity toxicity to lepidoptera pest, and other Bt gene then has single-minded toxicity to insects such as Diptera or Coleopteras.After the Bt gene changes crop over to through the biotechnology means, can express toxalbumin and make plant possess insect-resistance with insecticidal activity.At present, the domestic and international crop that has obtained trans Bt gene has cotton, paddy rice, corn, wheat and rape etc.

Though the test kit that has the many Bt of can be used for transgenic proteins to measure on the market now; Different processes is expressed, the separation and purification raw material but different manufacturers possibly adopt; And diverse ways is confirmed the value of reference material; And protein detection reference material and magnitude tracing transmit the famine of system and make that the measurement standard of upper level can not be traceable to reference material in institute in the different test kits, but also just can't guarantee accuracy and the traceability of different test kits institute with the reference material value, cause the inaccurate and shortage comparability of mensuration result; Supervision brings hidden danger to Biosafety, produces economy and social concerns such as a series of laws, trade thus.Therefore; In China's scope, develop Bt protein standard material as early as possible, set up sensitive and accurate method for quantitatively determining and Bt transgenic plant albumen magnitude tracing and transmit system; For the traceability, accuracy and the comparability that will guarantee to measure the result from now on, seem very urgent and significant.

In recent years, Along with people's found to the further investigation of debaryomyces hansenii (Hansenula polymorpha) biological characteristics that its advantage as the exogenous gene expression host manifested gradually.Debaryomyces hansenii also is a kind of methyl alcohol nutritional type yeast, and is similar with pichia spp.The metabolic main path of methyl alcohol has two kinds (Lodeboer A.M., et al., 1985) in the debaryomyces hansenii: a kind of is in peroxysome, to carry out, and (methanol oxidase, MOX) effect generates formaldehyde and H down to methyl alcohol at methanol oxidase ₂O ₂, formaldehyde is again through formaldehyde dehydrogenase (formaldehyde dehydrogenase) and hydrogenlyase (formate dehydrogenase, FMD) generation CO under the effect ₂, H ₂O ₂(catalase, effect CAT) generates H down at katalase ₂O and O ₂It is external that another pathways metabolism of methyl alcohol occurs in px; Methyl alcohol katalysis through a series of enzymes in tenuigenin changes carbohydrate at last into; Wherein (dihydroxyacetone synthase DHAS) is the key enzyme of this process to the Protosol synthetic enzyme.Various key enzymes in the methyl alcohol metabolic process; The expression that comprises MOX, DHAS and CAT etc. is all mediated at transcriptional level, and they receive inducing of methyl alcohol, glycerine and sorbyl alcohol, checked by glucose and alcoholic acid; But when glucose concn was lower than 0.1%, sun was held back effect and promptly is disengaged.Under the complete inductive condition of methyl alcohol; Peroxysome can account for 80% of cell TV; MOX and DHAS can account for 15% of total protein of cell, and their promotor has the function of extremely strong startup downstream gene expression, have been used as the promotor commonly used that foreign gene is expressed in yeast at present.Inducible promoter is widely used in expression of exogenous gene with its strong startup function and tight regulatory mechanism, and existing a lot of foreign proteins are being expressed in the debaryomyces hansenii cell under the regulation and control of these promotors efficiently.The inducible promoter that is used for debaryomyces hansenii exogenous protein expression system at present all need competence exertion effect under the condition that doses methyl alcohol exists; But because methyl alcohol is toxic substance; And inflammable and explosive, so just limited of the application of this system in fields such as medicine or food.Therefore; Various constitutive promoters just arise at the historic moment; The constitutive promoter that has been successfully applied to debaryomyces hansenii exogenous protein expression system has plasma membrane ATPase promotor (pPMA1) and translation elongation factor 1a promotor (TEF1) etc.; The application of these constitutive promoters not only need not in fermention medium, to add methyl alcohol, and has simplified zymotechnique and reduced cost etc.

Debaryomyces hansenii adopts auxotrophic strain to carry out the preliminary screening of recon as the exogenous protein expression host more; Obtained the multiple nutrients deficient strain at present; Like (Seon AhCheon, et al., 2009) such as leucine auxotrophy type, methionine(Met) defective type and tyrosine defective typies.Simultaneously, because debaryomyces hansenii is responsive to aminoglycoside antibiotics, antibiotics resistance genes such as G418, Zeocin and HYG also are widely used as selection markers.

During as the expression of exogenous gene host, foreign DNA is incorporated in the yeast genes group mainly can be divided into random integration and homologous recombination, must contain the homologous sequence of debaryomyces hansenii among the DNA that wherein homologous recombination need import with debaryomyces hansenii.Certain segment DNA sequence is consistent wholly or in part in homologous sequence and the yeast genes group, thereby causes foreign DNA can be incorporated in the yeast genes group through the homology exchange in this position.Debaryomyces hansenii transforms homologous sequence commonly used and comprises various strong promoters and rDNA (Klabunde J., et al., 2003) sequence, and the present invention has adopted debaryomyces hansenii plasma membrane ATPase gene as the homology integration sequence first.Fungi plasma membrane ATPase is that the seventies in last century is not found in Neuraspora crassa (Neurospora crassa) first, from schizosaccharomyces pombe (Schizosaccharomyces pombe), yeast saccharomyces cerevisiae (Saccharomycescerevisiae) and Neuraspora crassa (Neurospora crassa), obtains respectively subsequently purifying.Plasma membrane ATPase is the maximum albumen of content in the yeast plasma membrane; Account for 15% of total plasmalemma protein, account for 0.3% of total cell protein, its main effect is the proton pump of striding film as; Keep intracellular pH value and electrochemical gradient, thereby make the multiple nutrients material be able to transportation.Debaryomyces hansenii plasma membrane ATPase (Helen Cox, 2000) full length gene 2697bp, 898 amino acid of encoding.Be inserted in the expressed by Hansenula yeast carrier homologous sequence among the present invention first as exogenous origin gene integrator.

As the expression of exogenous gene host, debaryomyces hansenii has many advantages: 1) optimum growth temperature of debaryomyces hansenii is at 37～43 ℃, and thalli growth speed is fast, and fermentation time is short; 2) with the homologous sequence of rDNA as integration, can obtain the transformant of high copy, might greatly improve external source Recombinant Protein Expression amount; 3) a plurality of foreign genes can be integrated in the yeast genes group through single stage method simultaneously, and express at the same time or separately according to the doses relation.Above-mentioned advantage develops the expressed by Hansenula yeast system rapidly to become one of eukaryotic expression system of tool glamour, at present, has had multiple foreign protein genes in debaryomyces hansenii, to obtain expression, sees table 1:

The part foreign protein that table 1 is expressed in debaryomyces hansenii

*: BFSH α is a Bovine follicle-stimulation homone α subunit, and ScCnel is Sacchromyces cerevisiae calnexin

Before the present invention, also do not adopt the Bt gene after codon optimized in debaryomyces hansenii, to express this proteic report with the composing type mode, also nobody uses a compound enhancer sequence to improve the expression amount of Bt in the debaryomyces hansenii cell greatly.The codon that the present invention has a preference for according to yeast first; Synthetic a complete Bt antigen-4 fusion protein gene and a compound enhancer sequence; And utilization clone's the pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor (GAPDH) and the plasma membrane ATPase gene of debaryomyces hansenii strains A 16; Brand-new, debaryomyces hansenii efficient expression vector composing type and secretor type have been made up; After this foreign gene is imported the debaryomyces hansenii cell,, obtained to efficiently express Bt recombination fusion protein Yeast engineering bacterium strain through methods such as resistance screening, active detections; Again through the sequence of operations such as high density fermentation cultivation, nanofiltration and affinity chromatography of specified conditions, the reorganization Bt recombination fusion protein of final preparation can be used as protein reference material alternatives and is applied to the protein fields such as research of tracing to the source.

Summary of the invention

The technical problem that (one) will solve

One of the object of the invention is to confirm that a compound enhanser can improve the biological expression amount of Bt recombination fusion protein gene in the debaryomyces hansenii cell after codon optimized greatly.According to a preferential embodiment; The Bt antigen-4 fusion protein gene is at first optimized by the codon of having a preference for according to yeast and synthetic comes out; It is placed under a pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor (GAPDH) manipulation then, connects same compound enhanser, is integrated into together in the debaryomyces hansenii genome; Along with the growth of recombination yeast engineering bacteria, the Bt antigen-4 fusion protein gene can be efficiently expressed with the composing type mode under the effect of compound enhanser.

Two of the object of the invention provides the righttest reorganization debaryomyces hansenii growth and expression condition and target protein purification process fast.According to a preferential embodiment; The righttest grown cultures condition and the exogenous protein expression condition of relevant reorganization debaryomyces hansenii are provided; Composition, inoculum size, pH value, dissolved oxygen amount and incubation time etc. like substratum make the increment of this recombination yeast engineering bacteria and the secretory volume of recombinant protein all reach maximization as much as possible thus.From fermented liquid, obtain highly purified Bt external source recombinant protein through operations such as bactofugation, nanofiltration, affinity chromatographys, be applied to the protein fields such as research of tracing to the source so that it can be used as protein reference material alternatives.

(2) technical scheme

The present invention utilizes the expressed by Hansenula yeast system to express and produce the Bt recombination fusion protein with the composing type mode, and this recombinant protein has structure and the composition similar with native protein.According to a preferential embodiment, the invention provides and use the method for an enhanser to realize that the Bt antigen-4 fusion protein gene efficiently expresses and produces with the composing type mode in debaryomyces hansenii, this reorganization Bt has and similar structure and the composition of natural B t albumen.But the present invention simultaneously also provides Bt the method for high density fermentation production and this recombinant protein sharp separation and purifying in debaryomyces hansenii.

The present invention utilizes the expressed by Hansenula yeast system expression and produces the Bt recombination fusion protein, if with it through highly purified, can be used as protein reference material alternatives and be applied to the protein fields such as research of tracing to the source.

The Bt recombination fusion protein of indication of the present invention is expressed with the composing type mode in debaryomyces hansenii and is produced, and at first is that the proteic gene of coding Bt is optimized by the codon of having a preference for according to yeast and synthetic comes out; Meanwhile; Highly purified for later this target protein; Added one 6 histidine-tagged structure (6xHis) encoding sequence at Bt gene end; Then, this antigen-4 fusion protein gene is inserted on the Yeast expression carrier that has α-factor secreting signal peptide nucleotide coding sequence so that form a new antigen-4 fusion protein gene again.The effect of α-factor secreting signal peptide is the secretion of reorganization Bt albumen outside the debaryomyces hansenii born of the same parents of handling external source.On this plasmid vector, contain a constitutive promoter, this promotor is positioned at the upper reaches of the antigen-4 fusion protein gene of α-factor secreting signal peptide and Bt formation, and its handles fusion gene expression in the debaryomyces hansenii cell.In the downstream of fusion gene, contain a compound enhanser, it can greatly improve the expression amount of Bt recombination fusion protein.In this plasmid vector, also contain debaryomyces hansenii plasma membrane ATPase gene, played the effect of homologous sequence in its process in plasmid vector is incorporated into the yeast genes group.This carrier is after linearization process; Can be directed in the debaryomyces hansenii cell through methods such as electricity fusion or lithium chlorides; And then passing through the homologous recombination stable integration in the yeast chromosomal genome, the Bt recombination fusion protein that this recombination yeast is expressed in the fermentation culture process is able to be secreted in the substratum outside the born of the same parents under the guiding of α-factor secreting signal peptide.

The invention provides the method that greatly improves Bt recombination fusion protein biological yield in debaryomyces hansenii; According to a preferential embodiment; The invention provides and use a compound enhanser greatly to improve the method for Bt gene expression efficiency in debaryomyces hansenii; And this recombinant protein that the conventional expression system of this recombinant protein and native protein or other is produced is similar, can be used as protein reference material alternatives and is applied to the protein fields such as research of tracing to the source.

In the debaryomyces hansenii cell; The height of Bt antigen-4 fusion protein gene expression efficiency; The height of the extraction of this recombinant protein after being directly connected to, processing and utilization and the related prods cost of producing; According to a preferential embodiment, for improving the expression amount of Bt recombination fusion protein in debaryomyces hansenii, the gene of coding Bt recombinant protein can be optimized according to the codon that yeast is had a preference for; So that make this foreign gene more stable in the debaryomyces hansenii cell, efficient is higher when being transcribed into mRNA and translating into albumen.Simultaneously, with debaryomyces hansenii plasma membrane ATPase gene as homologous sequence, can be with exogenous gene high-efficient and stably being incorporated in the yeast genes group, thus obtain can stably express Bt recombinant protein the debaryomyces hansenii engineering strain.

The purity of Bt recombinant protein is directly connected to this Application of Recombinant scope in the leavened prod; According to a preferential embodiment; For fast purifying Bt histone, can at first use the dam filtration unit of molecular weight of difference that the yeast fermentation broth supernatant is carried out pre-treatment, and then through nickel post affinity chromatography; Each link in purge process; All can use the SDS-PAGE electrophoresis to detect, finally can obtain purity through aforesaid method and reach 99.9% Bt recombinant protein, can be used as protein reference material alternatives and be applied to the protein fields such as research of tracing to the source.

To the relevant item with other of the method used in the present invention structure of some plasmid vectors very importantly, they play an important role in the processed and applied of debaryomyces hansenii cell that obtains indication of the present invention and recombinant products thereof.The plasmid vector that is used for transformed yeast cell includes gene after codon optimized, coding Bt and handles this gene or a constitutive promoter and a compound enhanser that dna sequence dna is expressed at said yeast cell are formed.In some concrete embodiment, plasmid vector also comprises a selectivity or marker gene, is mainly used in the screening and the detection of reorganization debaryomyces hansenii cell.In some specific embodiments; Constitutive promoter of the present invention is a yeast glyceraldehyde-3-phosphate dehydrogenase promotor (GAPDH); As mentioned above; According to some concrete embodiment; The clone obtains the glyceraldehyde-3-phosphate dehydrogenase promoter sequence from pichia spp, be inserted into then among the plasmid expression vector pPIC9K and the replacement vector original position on inducible promoter-alcohol oxidase promoter so that make this plasmid expression vector express foreign protein with the composing type mode.According to some concrete embodiment; Plasmid expression vector of the present invention is used to transform debaryomyces hansenii; Its coded foreign protein is a reorganization Bt insecticidal proteins; Say that further this protein that the conventional expression system of reorganization Bt insecticidal proteins that this plasmid expression vector is coded and native protein or other is produced seemingly can be used as protein reference material alternatives and is applied to the protein fields such as research of tracing to the source.

The present invention also provides debaryomyces hansenii cytogenetics to transform and the method for yeast recombinant screen, and it may further comprise the steps: the codon of 1) having a preference for according to yeast, carry out the synthetic of Bt antigen-4 fusion protein gene (the PROTEIN C end has added the 6xHis label); 2) synthetic of the compound enhanser of TYMV-HCMV; 3) clone of pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor; 4) clone of debaryomyces hansenii plasma membrane ATPase gene; 5) make up a plasmid expression vector; The gene order that wherein contains coding Bt recombination fusion protein; This antigen-4 fusion protein gene at first is spliced to form a new antigen-4 fusion protein gene with α-factor secreting signal peptide nucleotide coding sequence each other, and they are placed under the control of pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor then.In this plasmid vector, contain a compound enhancer sequence, it can greatly improve the expression efficiency of Bt antigen-4 fusion protein gene.Meanwhile, also include in this plasmid expression vector and be used for the homologous sequence of exogenous origin gene integrator to the yeast genes group, this sequence can be PMA1 or other homologous sequence; 6) through electro fusion method or other method for transformation; Plasmid expression vector DNA after the above-mentioned linearizing is imported in the yeast cell; And the pichia spp cell after will transforming coats the RDB solid plate and selects on the substratum, and the yeast list bacterium colony that can on this substratum, grow is exactly the yeast recon that contains foreign gene; 7) select the yeast cell list bacterium colony that bears again; Be inoculated into one by one on the YPD solid plate substratum that contains different concns G418 (concentration be followed successively by 0.5,1.0,1.5,2.0mg/mL); Those the yeast list bacterium colonies that can on high density G418 flat board, grow have higher copy number of foreign gene, might have higher exogenous protein expression level.

According to a preferential embodiment, the invention provides through the reorganization debaryomyces hansenii incubation growth condition of optimization and the method for quickly purifying of Bt recombination fusion protein, it comprises following two stages: 1) yeast culture and albumen great expression stage.Inoculum size with 10% (accounting for the ratio of basic fermention medium volume) inoculation yeast engineering bacteria; Through 24～72 hours cultivation, the weight in wet base of yeast thalline will reach about 180～190g/L, in this culturing process, constantly add carbon source; And keep certain pH value and dissolved oxygen amount; Make the thalline high density fermentation, in the yeast bulk-growth, reorganization Bt insecticidal protein gene is efficiently expressed; 2) albumen fast purifying.Fermented liquid is handled through the membrane filtration of centrifugal and twice different size, handles through a nickel post affinity chromatography more at last, and the purity of Bt recombinant protein can reach more than 99.9%.

The present invention only mentions debaryomyces hansenii A16 bacterial strain when introducing yeast expression system in detail; Yet; As the yeast expression system that the expert of the art knew already, many primary yeast expression systems can utilize method provided by the present invention to carry out genetic transformation, expression and production.Therefore, all these yeast expression systems all should be included within the claim scope of the present invention.

Just as in the following instance that kind that will describe in detail; Used plasmid vector is an integrated plasmid expression system in the yeast conversion method that the present invention describes; According to a preferential embodiment; Plasmid expression vector used in the present invention is one and obtains improved pPIC9K; Its original AOX1 inducible promoter is replaced by pichia spp glyceraldehyde-3-phosphate dehydrogenase constitutive promoter, in addition, has also added a compound enhancer sequence greatly to improve the expression efficiency of foreign gene in debaryomyces hansenii.In this plasmid expression vector, also include simultaneously and be used for the homologous sequence of exogenous origin gene integrator in the yeast genes group, this sequence can be PMA1 or other homologous sequences.

The present invention includes following integral part: the codon of 1) having a preference for according to yeast; Being come out of Bt antigen-4 fusion protein gene by synthetic; This dna sequence dna is inserted in the T site of pEASY-T1 plasmid, the middle plasmid vector DNA transformed into escherichia coli DH5 α competent cell with obtaining is duplicated intermediate carrier; Carry out dna sequencing then, analyze the exactness and the integrity of the foreign gene that inserts; 2) according to known array; TYMV+HCMV is compound, and enhancer sequence is synthesized out; This dna sequence dna is inserted in the T site of pEASY-T1 plasmid, and the middle plasmid vector DNA transformed into escherichia coli DH5 α competent cell with obtaining is duplicated intermediate carrier; Carry out dna sequencing then, analyze the exactness and the integrity of the dna fragmentation that inserts; 3) according to known array (GenBank:U62648.1), the primer at synthetic two ends is a template with the pichia spp genomic dna; Through pcr amplification, can obtain pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor, this dna sequence dna is inserted in the T site of pEASY-T1 plasmid; With the middle plasmid vector DNA transformed into escherichia coli DH5 α competent cell that obtains; Intermediate carrier is duplicated, carry out dna sequencing then, analyze the exactness and the integrity of the exogenous dna fragment that inserts; 4) utilize conventional Protocols in Molecular Biology (restriction endonuclease and ligase enzyme are handled); With pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor be inserted among the above-mentioned pPIC9K and the replacement vector original position on inducible promoter-alcohol oxidase promoter so that make this expression vector express foreign protein genes with the composing type mode; 5) according to known PMA1 gene order, the primer at synthetic two ends is a template with the debaryomyces hansenii genomic dna; Through pcr amplification; Obtain the PMA1 dna sequence dna, be inserted in the T site of pEASY-T1 plasmid, with the carrier transformed into escherichia coli DH5 α competent cell that obtains; Carrier is duplicated, then the exactness of sequencing analysis PMA1 gene and integrity; 6) utilize conventional Protocols in Molecular Biology (restriction endonuclease and ligase enzyme are handled); Be inserted in the corresponding restriction enzyme site in the pPIC9K plasmid Bt antigen-4 fusion protein gene, the compound enhancer sequence of TYMV-HCMV, PMA1 gene and pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor are directed respectively, form brand-new a, composing type and secretor type yeast plasmid expression vector thus; 7) screening of high expression level yeast recon; Merge or other genetic transforming method through electricity, the yeast plasmid expression vector after the above-mentioned reorganization (linearizing) is imported the yeast recipient cell, select to cultivate on the substratum at the RDB solid plate; After treating that yeast list bacterium colony occurs; Again it is transferred on the YPD solid plate substratum that contains different concns G418 one by one, can resists the yeast recon of high density G418 owing to have higher Bt copy number of foreign gene, thereby might have higher exogenous protein expression amount; Therefore, select the yeast recon that on the YPD of maximum concentration G418 solid plate substratum, to grow and carry out follow-up operation; 8) condition of the reorganization the righttest grown cultures of debaryomyces hansenii and expression is provided; 9) fermented supernatant fluid after steps such as centrifugal, filtration and nickel post affinity chromatography, can obtain purity and reach the Bt recombination fusion protein more than 99.9% respectively.

(3) beneficial effect

Adopt the method for the invention; Can effectively the Bt foreign gene after codon optimized be incorporated in the debaryomyces hansenii genome through the mode of PMA1 gene order with homologous recombination; The Yeast engineering bacterium strain that obtains can with the composing type mode stable with express the Bt recombination fusion protein efficiently; The fermentation and the purifying process of this recombinant protein are provided simultaneously, have been applicable to large-scale production Bt recombinant protein.

Description of drawings:

In order to describe some preferential embodiment of the present invention in detail, and for referencial use with corresponding drawing:

The compound enhanser of Fig. 1 TYMV-HCMV is formed synoptic diagram: curved portion is an EcoR I restriction enzyme site; The 9th～113 nucleotide base is derived from 3 ' the end non-coding region (single setting-out part) of TYMV; The 119th～609 nucleotide base is derived from the 201st～690 part (two setting-out part) of HCMV early gene enhanser; Dotted portion is a Not I restriction enzyme site.

Codon optimized and the transformation front and back contrast of Fig. 2 Bt toxoprotein gene: the gene order that the N representative is codon optimized and transformation is preceding; M represents codon optimized and improved gene order;

The structure whole process of Fig. 3 composing type yeast efficient expression vector EBt-ATP-GPIC9K and WBt-ATP-GPIC9K;

Fig. 4 SDS-PAGE electrophoresis detection Bt fusion protein expression situation: M is standard molecular weight albumen (Blue Plus Proteinmarker 16～94KDa, Beijing TransGen Biotech Company products); The negative contrast of CK-, the restructuring yeast strains that GPIC9K transforms is cultivated 72 hours fermented liquid supernatant; 1～4 is derived from the different recons of WBt-ATP-GPIC9K transformed yeast respectively cultivates 72 hours fermented supernatant fluids; 5～8 are derived from EBt-ATP-GPIC9K transformed yeast recon respectively cultivates 72 hours fermented supernatant fluids, and the molecular weight of Bt recombination fusion protein is about 68.5kD;

Bt Recombinant Protein Expression situation and the Bt recombination fusion protein after the affinity chromatography under the different incubation times of Fig. 5 detect: M is standard molecular weight albumen (Blue Plus Protein marker 16～94KDa, Beijing TransGen Biotech Company products); 1～4 swimming lane incubation time is respectively the fermented supernatant fluid of 12h, 24h, 48h and 72h; 5 is the Bt recombinant protein after the nickel post affinity chromatography.

The ELISA method of using Fig. 6 detects different yeast recon Bt expression of recombinant proteins situation: A1～3, B1～3 are EBt-ATP-GPIC9K transformed yeast recon fermented supernatant fluid; C1～3 and D1～3 are WBt-ATP-GPIC9K transformed yeast recon supernatant; The positive contrast of E1; The negative contrast in E2～3.

Embodiment

Be example with debaryomyces hansenii constitutive expression Bt recombination fusion protein below, describe some preferential embodiments, but application of the present invention be not limited only to this.Just some the special preferential embodiments that further describe of the present invention are according to the patented claim requirement and in order to explain and explain the content of this patent.Obviously, do not deviating within the spirit and scope of the present invention, can do further improvement and variation on this basis.

The synthetic of the compound enhanser of embodiment 1:TYMV-HCMV

According to the Turnip yellowmosaic virus (TYMV) that had announced already (see GenBank, accession number: X07441) and Human cytomegalovirus (HCMV) (see GenBank; Accession number: dna sequence dna K03104); The codon of having a preference for according to yeast (Sharp P M, et al., 1986); Artificial design and synthesized a new compound enhancer sequence (seeing SEQ ID NO:1); In this sequence, it is 3 ' the end non-coding region (genomic the 6214th～6318 nucleotide base of TYMV) (seeing single setting-out part in the accompanying drawing 1) of TYMV that 105 nucleotide bases are arranged, and two setting-out parts of accompanying drawing 1 (are seen in the core (the 201st～690 nucleotide base) that has 490 nucleotide bases to be derived from the early gene enhanser of HCMV; Meanwhile; For ease of the structure of Yeast expression carrier after this, in the process of this compound enhancer sequence of synthetic, synthetic and added restriction enzyme site EcoR I at 5 ' end of this dna sequence dna; Added restriction enzyme site Not I (the synthetic and splicing work of above-mentioned DNA by Shanghai Bo Ya biotech company on behalf of accomplishing) at 3 ' end of this dna sequence dna.

Embodiment 2: the synthetic of the Bt gene after codon optimized

The Bt gene comes from bacillus thuringiensis at first; Its codon is a prokaryotic organism institute preference; And debaryomyces hansenii belongs to eukaryote; So they are existing certain difference aspect the gene codon preference, and this species diversity has influence on Bt gene and stability and the expression efficiency of transcription product in the debaryomyces hansenii cell thereof possibly.For improving the biological yield of Bt, (see GenBank, accession number: AR110596) according to the dna sequence dna (being the codon of plant preference originally) and the aminoacid sequence of the Bt gene of having announced already; Under the prerequisite that does not change its aminoacid sequence, (see SEQ ID NO:2), the codon (Sharp P M, the et al. that have a preference for according to yeast; 1986), artificial design and the dna encoding sequence of having synthesized new Bt maturation protein are compared before codon optimized and improved Bt gene and the transformation; 422 nucleotide bases have wherein been changed; Relate to 340 codons altogether, and that G+C content becomes by original 48% is present 35%, accompanying drawing 2 is seen in contrast before and after the genetic modification.At the same time, since in order to facilitate the construction of yeast expression vector and purification of recombinant proteins in Bt synthetic nucleotide coding sequence of the process, the gene at the 5 'end of the first translation initiation codon ATG before synthesis and adding the restriction enzyme sites Xho? I gene product and yeast Kex2 cutting recognition sequence

(see SEQ? ID? NO: 3); in the gene 3 'end prior to the termination codon TAA structure coding increases the 6xHis tag sequence

TAA, after the addition of two restriction sites EcoR? I and Not? I (synthesis and splicing the genes from the previous work on behalf completed Boya Biotechnology).

The clone of compound enhanser of embodiment 3:TYMV-HCMV and Bt gene

The compound enhancer sequence of TYMV-HCMV and the Bt antigen-4 fusion protein gene dna fragmentation of above-mentioned synthetic directly are inserted into respectively in the T site in pEASY-T1 (available from Beijing TransGenic company) plasmid; According to the method that the said firm provided; The bacterial clone (seeing accompanying drawing 3) of plasmid vector TYMV-HCMV-T and Bt-T in the middle of obtaining containing; Then; Through dna sequencing, confirm that compound enhanser of its contained TYMV-HCMV-T and Bt-T gene order are correct and complete (dna sequencing are accomplished by Beijing Biokit, Inc.).

Embodiment 4: the clone of pichia spp glyceraldehyde-3-phosphate dehydrogenase promotor

According to known pichia spp glyceraldehyde-3-phosphate dehydrogenase promoter sequence (GenBank:U62648.1), synthetic respectively primers F and the R that is positioned at the promotor two ends, wherein F be 5 '-AT GGATCCTTTTTTGTAGAAATGTCTTGGTGTC-C-3 ' (line part be BamH I point of contact), R be 5 '-AT GAGCTCTGTGTTTTGATAGTTGTTCAATTGATTG-3 ' (the line part is Sac I point of contact) is template (the genome process for extracting is referring to " the molecular cloning experiment guide third edition " 485 pages) with the pichia spp genomic dna, is primer with F and R; Through PCR, amplification obtains glyceraldehyde-3-phosphate dehydrogenase promoter sequence (GAPDH), and amplified fragments directly is inserted in the T site in the pEASY-T1 plasmid; According to the method that the said firm provided; The bacterial clone (seeing accompanying drawing 3) of plasmid vector GAPDH-T in the middle of obtaining containing, then, through the nucleotide sequencing analysis; Confirm that GAPDH is correct and complete, sees SEQ ID NO:4.

Embodiment 5: the clone of debaryomyces hansenii plasma membrane ATP enzyme gene

According to known debaryomyces hansenii plasma membrane ATP enzyme gene nucleotide series (seeing GeneBank:AF109913), design respectively and synthetic primer primer1 and primer2, wherein the Primer1 sequence is: 5-AT CATATGATGCTGCAACTGAACCAACCAAGGAG-3 ', the Primer2 sequence is: 5 '-AT GCATGCTCAGTTGGACTTCTCGTGCTGAGTAGAG-3 '.Primer1 and primer2 are added with Nde I (CATATG) respectively and Sph I (GCATGC) restriction enzyme is cut the site, is used for the structure of follow-up Yeast expression carrier.With the debaryomyces hansenii genomic dna is template, pcr amplification debaryomyces hansenii plasma membrane ATP enzyme gene, and pcr amplification product carries out 1% agarose gel electrophoresis; Cutting purpose band also reclaims test kit with sepharose and reclaims target DNA fragment; Be directly connected to then (available from TransGenic company) in the T site in the pEASY-T1 plasmid,, obtain containing the bacterial clone ATP-T (seeing accompanying drawing 3) of plasma membrane ATP enzyme gene according to the method that the said firm provided; Then; Through the nucleotide sequencing analysis, confirm that plasma membrane ATP enzyme gene is correct, see SEQ ID NO:5.

Embodiment 6: the structure of expression vector EBt-ATP-GPIC9K and WBt-ATP-GPIC9K

Carry out double digestion with restriction enzyme BamH I and Sac I, the GAPDH dna fragmentation among the plasmid vector GAPDH-T in the middle of being connected is scaled off, separate and reclaim this dna fragmentation through agarose gel electrophoresis.Handle plasmid pPIC9K (American I nvitrogen Company products) with same restriction enzyme; Through the pPIC9K DNA after agarose gel electrophoresis separation and the recovery linearizing; Link together after then above-mentioned two dna fragmentations being mixed and with ligase enzyme and just obtain middle plasmid vector GPIC9K (seeing accompanying drawing 3), use above-mentioned plasmid vector transformed into escherichia coli cell DH5 α (available from U.S. GIBCO company) then conveniently to carry out duplicating and preserving of this plasmid.

Carry out double digestion with restriction enzyme Nde I and Sph I, the debaryomyces hansenii plasma membrane ATP enzyme gene DNA fragment among the plasmid vector ATP-T in the middle of being connected is scaled off, separate and reclaim this dna fragmentation through agarose gel electrophoresis.Handle plasmid GPIC9K with same restriction enzyme; Through the GPIC9K DNA after agarose gel electrophoresis separation and the recovery linearizing; Then with above-mentioned two dna fragmentations mix the back and with ligase enzyme link together just obtain in the middle of plasmid vector ATP-GPIC9K (seeing accompanying drawing 3), then with above-mentioned plasmid vector transformed into escherichia coli cell DH5 α conveniently to carry out duplicating and preserving of this plasmid.

Carry out double digestion with restriction enzyme Xho I and Not I, the Bt gene DNA fragment among the plasmid vector Bt-T in the middle of being inserted in is scaled off, separate and reclaim this dna fragmentation through agarose gel electrophoresis.Handle plasmid ATP-GPIC9K with same restriction enzyme; Through the ATP-GPIC9K DNA after agarose gel electrophoresis separation and the recovery linearizing; Just obtain expressed by Hansenula yeast carrier WBt-ATP-GPIC9K (seeing accompanying drawing 3) with linking together after above-mentioned two dna fragmentations mixing and with ligase enzyme; In this expression vector, do not contain the compound enhancer sequence of TYMV-HCMV, mainly as negative control with the enhancement of explanation TYMV-HCMV compound enhanser to the Bt exogenous gene expression.With above-mentioned plasmid vector transformed into escherichia coli cell DH5 α (available from U.S. GIBCO company) conveniently to carry out duplicating and preserving of this plasmid.

Carry out double digestion with restriction enzyme Xho I and EcoR I, the Bt gene DNA fragment among the plasmid vector Bt-T in the middle of being inserted in is scaled off; Carry out double digestion with EcoR I and Not I, the compound enhancer DNA sequence of TYMV-HCMV is scaled off on middle plasmid vector TYMV-HCMV-T, separate respectively and reclaim above-mentioned dna fragmentation through agarose gel electrophoresis.Handle plasmid ATP-GPIC9K with same restriction enzyme; Through the ATP-GPIC9K DNA after agarose gel electrophoresis separation and the recovery linearizing; Just obtain expressed by Hansenula yeast carrier EBt-ATP-GPIC9K (seeing accompanying drawing 3) with linking together after above-mentioned three dna fragmentations mixing and with ligase enzyme; In this expression vector, contain the compound enhancer sequence of TYMV-HCMV, use above-mentioned plasmid vector transformed into escherichia coli cell DH5 α (available from U.S. GIBCO company) then conveniently to carry out duplicating and preserving of this plasmid.

Plasmid vector pPIC9K is available from American I nvitrogen company, and it is a methanol evoked expression plasmid carrier, contains an inducible promoter-alcohol oxidase promotor (AOX1), and under the inducing of methyl alcohol, efficiently expressing of foreign gene inserted in adjustable downstream.Before this expression vector MCS, contain α-factor secreting signal peptide nucleotide coding sequence, with the foreign gene amalgamation and expression after, can guide the external source recombinant protein to the yeast cell external secretion.In the process outside being secreted into born of the same parents, this signal peptide can be cut down by yeast Kex2 gene expression product, thereby does not change the N terminal sequence of recombinant protein.

Embodiment 7: the preparation of linearization plasmid expression vector dna

At first use alkaline lysis (referring to the molecular cloning test guide); Extract EBt-ATP-GPIC9K and WBt-ATP-GPIC9K DNA from the medium and small preparation respectively of above-mentioned e.colidh5; Carrying out enzyme with 1～2 times of excessive restriction enzyme Xba I then cuts; Make it complete linearizing, whether agarose gel electrophoresis capable of using detects enzyme and cuts complete.Then with phenol and chloroform respectively the above-mentioned enzyme of extracting cut product, ethanol sedimentation is abandoned supernatant, collecting precipitation after lyophilize, is dissolved in deposition in the aseptic deionized water again ,-20 ℃ of preservations are subsequent use.

Embodiment 8: the genetic transformation of yeast cell

The debaryomyces hansenii bacterium liquid (strains A 16 is so kind as to give by professor Li Ying of China Agricultural University) of-72 ℃ of preservations is inoculated into (1% yeast extract among the 5mL YPD; 2% Tryptones; 2% glucose); 37 ℃ of concussions were cultivated about 1 day, and cultured bacterium liquid is re-seeded among the 100mL YPD with 1% inoculum size, and 37 ℃ of concussion incubated overnight (8h) are to OD ₆₀₀=1.3～1.5,4000 rev/mins centrifugal 5 minutes, outwell supernatant, sedimentary yeast cell is resuspended in 40mL solution a (50mM potassium phosphate buffer; PH7.5,25mM DTT) in, 37 ℃ of temperature are bathed 15min; 4000 rev/mins centrifugal 5 minutes, outwell supernatant, sedimentary yeast cell is resuspended in solution b (the 270mM sucrose of 200mL ice precooling; 10mMTris-HCl, pH7.5,1mM MgCl ₂) in, 4000 rev/mins centrifugal 5 minutes, outwell supernatant; Sedimentary yeast cell is resuspended among the solution b of 100mL precooling; 4000 rev/mins centrifugal 5 minutes, outwell supernatant, sedimentary yeast cell is resuspended among the solution b of 1mL precooling; Draw 80uL in the 1.5mL centrifuge tube; (4～5ug) abundant mixings are respectively transferred in the aseptic electric shock cup of 0.2cm behind the ice bath then, utilize the appearance PrecisionPulse that shocks by electricity with above-mentioned linearizing expression vector EBt-ATP-GPIC9K and WBt-ATP-GPIC9K DNA ^TM(BTX Company products) imports linearizing expression vector DNA in the yeast competent cell, and the shock parameters of using is voltage 1.5kV, electric capacity 50uF, resistance 125 Ω.After electric shock is accomplished, in the electric shock cup, add YPD substratum (1% yeast extract, 2% casein peptone of 1mL room temperature immediately; 2% glucose), fully behind the mixing, 37 ℃ left standstill 1 hour; Coat solid YPD substratum then and (added 2% agar powder in the liquid nutrient medium; 0.2mg/mL G418), flat board was inverted in 37 ℃ of constant incubators 2～3 days, occurred to transforming recon.

Embodiment 9: the screening of high expression level yeast strain

Picking is to the YPD substratum that contains gradient G 418 (containing 0.5mg/mL, 1mg/mL, 1.5mg/mL, 2mg/mL G418 respectively) one by one with aseptic toothpick for the yeast list bacterium colony (transformant) that will go up growth at YPD substratum (0.2mg/mL G418), and flat board was inverted in 37 ℃ of constant incubators 1～2 day.Along with the exogenous plasmid that carries the G418 resistant gene is incorporated into the increase of the copy number in the yeast genes group, transformant strengthens the resistance of G418.Picking can contain the transformant of growing on the YPD flat board of 2mg/mL G418, is inoculated in 100mL BMGY substratum [1% yeast extract, 2% casein peptone; 100mM potassium phosphate buffer (pH7.0), 1.34%YNB, 0.00004%Biotin; 1% glycerine (v/v)] in, 37 ℃ of concussions were cultivated 4 days, and 10000 rev/mins of fermented liquids are centrifugal 5 minutes; Collection possibly contain the proteic supernatant of recombinant C P4-EPSPS, carries out the SDS-PAGE electrophoresis detection.

Can know from accompanying drawing 4; In the restructuring yeast strains culture supernatant liquid swimming lane that is obtained through expression vector EBt-ATP-GPIC9K conversion, contain Bt target protein band, in the restructuring yeast strains culture supernatant liquid swimming lane that is obtained through expression vector WBt-ATP-GPIC9K conversion, then do not having Bt target protein band (detecting after cultivating 72h).This result shows: the compound enhanser of TYMV-HCMV has played keying action aspect the expression amount of raising Bt antigen-4 fusion protein gene in debaryomyces hansenii.

The yeast transformant that can on the YPD of 2mg/mL G418 flat board, grow of picking part at random, fermentation culture is got fermented liquid supernatant and is carried out ELISA and detect.Bt ELISA detection kit is available from U.S. Agdia company; According to the method that test kit provided; Get respectively in each aperture that 100 microlitre fermented supernatant fluids add the enzyme chain reaction plate to, after wash, add enzyme labelled antibody, TMB chromogenic substrate successively, under the wavelength of 650nm, measure absorbancy.Measure the result and find that all 6 EBt-ATP-GPIC9K that can on the YPD of 2mg/mL G418 flat board, grow transform recon and can both produce positive reaction, and difference is not obvious.Therefrom random choose goes out a strain and preserves as engineering bacteria, and with its called after HP-Bt.And all can not produce positive reaction (seeing accompanying drawing 5) in that 6 yeast recons that transform through expression vector WBt-ATP-GPIC9K are the same with negative control.

Embodiment 10: the high density fermentation of recombination microzyme

1, the preparation of seed liquor

Picking list bacterium colony is inoculated in restructuring yeast strains HP-Bt in the 10mL BMGY substratum, 37 ℃ of concussion overnight cultures; Transfer in 100mL BMGY substratum with 10% inoculum size, 37 ℃ of concussion overnight cultures are transferred in 1L BMGY substratum with 10% inoculum size again; 28 ℃ of concussion overnight cultures; It is transferred in the 3L BMGY substratum, and 37 ℃ of concussions were cultivated 2 days, as the seed liquor of high density fermentation.

2, the high density fermentation of recombination yeast in the 50L fermentor tank

This fermenting process can be divided into two stages: 1) first cultivation stage: in 50L fermentor tank (Zhenjiang Oriental Bio-engineering Technology Co., Ltd), held 30L basis fermention medium [10 * Basal Salts:2.67% phosphoric acid, 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% Pottasium Hydroxide+4% glycerine], adding ammoniacal liquor makes the pH value of this substratum maintain about 4.0 and (discovers that low pH can prevent the enzyme liberating of target protein earlier before inoculation; Be higher than at 5 o'clock at pH, the expression amount of target protein reduces), again according to following ratio; In every liter of basic fermention medium, add 4.37mL trace salt solution PTM1 (0.6% copper sulfate; 0.008% Soiodin, 0.3% manganous sulfate, 0.02% Sodium orthomolybdate; 0.002% boric acid; 0.05% NSC 51149,2% zinc chloride, 6.5% ferrous sulfate; 0.025% vitamin H, 0.5% sulfuric acid).Ratio in 10% (with the ratio of the volume of the basic fermention medium) seed liquor that inoculation prepares before this, 37 ℃ of aeration-agitations (rotating speed is 375 rev/mins) were cultivated about 24 hours.Carrying out in the process of this stage, along with the growth of yeast thalline, the dissolved oxygen amount in the substratum will reduce by 100% gradually, and after the carbon source in the substratum runs out of, dissolved oxygen amount will be increased to more than 80% once again, and the weight in wet base of thalline will reach 85～95g/L this moment.2) the albumen great expression stage (24～72h): from postvaccinal second day, through peristaltic pump flow feeding liquid, feed supplement liquid was 50% glycerine (wherein containing 12mL PTM1/L), stream dosage be 18mL/L/ days.37 ℃ of aeration-agitations are cultivated, and the weight in wet base of thalline will reach 170～180g/L behind the 48h.Along with the growth of thalline, the pH value reduces gradually, keeps the pH value about 4.0 with ammoniacal liquor, adjusts air flow simultaneously the dissolved oxygen amount in this stage is maintained more than 20% all the time.Whenever descended centrifugal 10 minutes for 4 ℃ through 5000rpm at a distance from 24 hours peek milliliter fermented liquids; Get 30uL fermented liquid supernatant liquid and carry out the SDS-PAGE detection; Discovery has the observable protein band of naked eyes, and molecular weight is about 68.5kDa, and is identical basically with the Bt recombinant protein molecular weight in inferring.In addition, find from electrophorogram that after cultivating 72 hours, Bt expression of recombinant proteins amount reaches climax (seeing accompanying drawing 6).

Embodiment 10: the purifying of recombinant protein

After treating that a fermentation period is all over, leave and take the 500mL fermented liquid directly carries out next round as seed liquor (inoculum size is 5%) fermenting process.Similar operation adds up to carry out 3 takes turns, and takes turns in the fermenting process every, all the increment and the Bt Recombinant Protein Expression amount of thalline is measured.In addition, take turns after fermenting process finishes fully, also get a little bacterium liquid and coat on the YPD solid plate every; And therefrom any 10 yeast lists of picking bacterium colony, its genome of rapid extraction (Cai Chuanqi etc., 2001) DNA; Carry out PCR and detect, the result finds that the living weight of thalline, the speed of growth and Recombinant Protein Expression amount are taken turns kept stable in the fermenting process at each; In addition, the detected result of the PCR debaryomyces hansenii bacterial strain that also confirms to recombinate has good genetic stability (seeing table 2).

The genetic stability of table 2.HP-Bt bacterial strain and the mensuration of exogenous protein expression stability

PCR is positive	The thalline weight in wet base (g/L) of growing 24 hours	Induce 72 hours Bt expression amounts (mg/L)
			100％	89	41
100％	91	43
			100％	90	41

After a fermentation period finished, except that staying the 500mL fermented liquid as the seed liquor, remaining fermented liquid was used for the purifying of Bt recombinant protein.Fermented liquid is left and taken supernatant through centrifugal 10 minutes of 4 ℃ of 5000rpm.Fermented liquid supernatant uses the molecular weight that dams to handle as the nf membrane of 10KDa (the bright utmost point in Shanghai Chemical Industry Science Co., Ltd, PIN: 2426538, method of use is seen the said firm's explanation), keeps phegma, and final volume is about 700mL.This phegma is carried out desalting treatment; In the 700mL phegma, add 6.3L zero(ppm) water; Be about to 10 times of phegma dilutions, then with diluent once more through the nanofiltration membrane treatment of above-mentioned 10KDa, the also corresponding dilution of salt ionic concentration is 10 times in the phegma that obtains at this moment; So repetitive operation is 5 times, i.e. salt ionic concentration dilution 10 in the phegma ⁵Doubly, finally obtain the 700mL phegma, after this phegma lyophilize, can obtain the recombinant protein lyophilized powder (purity is approximately about 13%) of preliminary purification.

Above-mentioned albumen lyophilized powder is suspended in again in the 20mM Tris-HCl damping fluid of 50mL (pH7.9 contains the 5mM imidazoles, 0.5M NaCl).Prepare the nickel post of 20mL, at first use aseptic washing post (10 times of column volumes), flow velocity 1mL/mim uses sample-loading buffer I (10mM Tris-HCl, pH7.9 contain 0.25M NaCl) to carry out balance (10 times of column volumes) then, and flow velocity is 1mL/min.The above-mentioned protein sample liquid of the 50mL speed with 1mL/min is joined in the pillar.Continue balance (10 column volumes) with sample-loading buffer I; Then respectively with the 10mMTris-HCl damping fluid (pH7.9 that contains 50mM, 100mM and 400mM imidazoles; Contain 0.25M NaCl) carry out wash-out, flow velocity is 2mL/min, collects the elution peak in each stage; Behind the SDS-PAGE electrophoresis, detect the Bt recombinant protein with UVP gel imaging appearance and reclaim purity.

In above-mentioned purifying flow process, all leave and take small amount of sample so that carry out the mensuration (seeing table 3) of total protein concentration, purity and the recovery after each operation steps.Detect proteic concentration with the Bradford method.

The pure article of the recombinant protein that finally makes carry out the SDS-PAGE electrophoresis; After coomassie brilliant blue staining and decolouring; Electrophorogram is analyzed through LabWork software (U.S. UVP Company products), and the result shows that Bt recombinant protein concentration reaches more than 99.9% and (see accompanying drawing 6) that the Recombinant Protein Expression amount is about 41mg/L.

The purifying of table 3.Bt recombinant protein

Step	TV (L)	Cycles of concentration	Total protein concentration (g)	Purity (%)	The recovery (%)
						Fermented liquid supernatant	?40	1	?12.5	～13.0％	100.0％
The 50kDa hollow fiber filter membrane	?39	1	?12.3	～13.0％	～99％
						The 10kDa nf membrane	?0.7	57	?10.5	～15.6％	～93％
Nickel post affinity chromatography	?0.01	4000	?0.9	～99.9％	～57％

Reference

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Claims

1. one kind is used compound enhanser to improve the method for Bt fusion rotein in the expressed by Hansenula yeast amount.

2. like the method in the claim 1, wherein said compound enhanser has the nucleotide sequence shown in 1 in the sequence table.

3. like the method in the claim 1, the dna sequence dna of coding Bt fusion rotein is characterized in that having the nucleotide sequence shown in the sequence table 3.

4. like the method in the claim 1, need to make up a kind of expression plasmid carrier, it is characterized in that it contains the dna sequence dna of the coding Bt fusion rotein described in described compound enhancer sequence of claim 2 and the claim 3.

5. the expression vector described in right 4 is characterized in that, when construction of expression vector, the initial vector of employing be pPIC9K or other can constitutive expression justacrine recombinant protein to the outer plasmid vector of born of the same parents.

6. like the method in the claim 1, need a kind of yeast host cell, it is characterized in that it comprises the expression vector described in the claim 4 and 5.

7. the host cell described in claim 6, it is meant the yeast strain that debaryomyces hansenii strains A 16 or other can expression alien genes.

8. the method for utilizing the described debaryomyces hansenii strains A 16 of claim 7 to efficiently express and produce the Bt recombination fusion protein, comprising four-stage:

1) seed liquor preparation: picking yeast list bacterium colony; Be inoculated in the 10mL YPD liquid nutrient medium, after 37 ℃ of shaking culture are spent the night, this culture transferred in 100mL YPD substratum; 37 ℃ of shaking culture are after 24 hours; This culture is transferred in 3L BMGY substratum, 37 ℃ of shaking culture 48 hours, then with it as seed liquor;

2) first cultivation stage: in the 20L fermentor tank, held 10L basis fermention medium [10 * Basal Salts:2.67% phosphoric acid, 0.093% calcium sulfate, 1.82% vitriolate of tartar, 1.49% sal epsom, 0.413% Pottasium Hydroxide+4% glycerine], before inoculation, added ammoniacal liquor earlier the pH value of this substratum is maintained about 4.0, again according to following ratio; In every liter of basic fermention medium, add 4.37mL trace salt solution PTM1 (0.6% copper sulfate, 0.008% Soiodin, 0.3% manganous sulfate; 0.02% Sodium orthomolybdate, 0.002% boric acid, 0.05% NSC 51149; 2% zinc chloride; 6.5% ferrous sulfate, 0.025% vitamin H, 0.5% sulfuric acid).In the seed liquor that the ratio inoculation that accounts for basic fermention medium volume 10% prepares before this, 37 ℃ of aeration-agitations (rotating speed maintains 375 rev/mins from start to finish) were cultivated about 24 hours;

3) the albumen great expression stage: from postvaccinal second day, through peristaltic pump flow feeding liquid, feed supplement liquid was 50% glycerine (wherein containing 12mL PTM1/L), and the stream dosage is 18mL/L/ days.37 ℃ of aeration-agitations are cultured to 72h, keep the pH value about 4.0 with ammoniacal liquor, adjust air flow simultaneously the dissolved oxygen amount in this stage is maintained more than 20% all the time.

4) fermented liquid is left and taken supernatant through centrifugal 10 minutes of 4 ℃ of 5000rpm.Use the molecular weight that dams to be the nanofiltration membrane treatment supernatant of 10KDa, keep phegma.With zero(ppm) water with 10 times of phegma dilutions, and then the nanofiltration membrane treatment through 10KDa, so repetitive operation is 5 times.After the phegma lyophilize, can obtain the Bt recombinant protein lyophilized powder of preliminary purification.It is suspended in the 20mMTris-HCl damping fluid of 50mL (pH7.9 contains the 5mM imidazoles, 0.5M NaCl) again.Prepare the nickel post of 20mL, at first use aseptic washing post (10 times of column volumes), flow velocity 1mL/mim uses sample-loading buffer I (10mM Tris-HCl, pH7.9 contain 0.25M NaCl) to carry out balance (10 times of column volumes) then, and flow velocity is 1mL/min.The speed of above-mentioned protein sample liquid with 1mL/min is joined in the pillar.Continue balance (10 column volumes) with sample-loading buffer I; Carry out wash-out with the 10mM Tris-HCl damping fluid (pH7.9 contains 0.25MNaCl) that contains 50mM, 100mM and 400mM imidazoles respectively then, flow velocity is 2mL/min; Collection contains the elutriant in Bt target protein stage, converges the back freeze-drying.

9. method as claimed in claim 8, wherein said Bt recombination fusion protein can be used as protein reference material alternatives after highly purified and are applied to the protein fields such as research of tracing to the source.