CN105695500A - Expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris - Google Patents
Expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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Abstract
The invention discloses an expression and purification method of chicken growth hormone recombinant protein in Pichia pastoris. The expression and purification method includes the steps of firstly, extracting total RNA (ribonucleic acid) from chicken pituitary, performing reverse transcription PCR (polymerase chain reaction) to obtain cDNA (complementary deoxyribonucleic acid) and conducting PCR by taking the cDNA as a template so as to obtain chicken growth hormone gene fragments; secondly, using the Pichia pastoris for constructing a recombinant expression vector pPIC9K-cGH; thirdly, subjecting Pichia pastoris GS115 to electrotransformation by the recombinant expression vector pPIC9K-cGH and screening to obtain a transgenic recombinant yeast strain GS115-pPIC9K-cGH; fourthly, obtaining the chicken growth hormone recombinant protein through methanol induced expression and purification, and identifying activity of the chicken growth hormone recombinant protein through cell activity test. In the expression and purification method of the chicken growth hormone recombinant protein in the Pichia pastoris, high-efficiency expression of the cGH (chicken growth hormone) recombinant protein in the Pichia pastoris is achieved and a purification system is established firstly, so that the high-purity chicken growth hormone recombinant protein with activity can be obtained. The expression and purification method of the chicken growth hormone recombinant protein in the Pichia pastoris is simple and convenient to operate, high in yield and low in cost and provides a basis for further studies on structures, functions and production application of chicken growth hormone.
Description
Technical field
The present invention relates to the production of recombiant protein in genetic engineering field and purification process, particularly to the chicken growth hormone recombiant protein high efficient expression in Pichia sp. and purification process。
Background technology
Chicken growth hormone (chickengrowthhormone, cGH) not only the normal growth of chicken is grown the adjustment effect playing necessity, and the multiple production traits of chicken is also had significant impact, such as body weight, feed conversion rate, lipopexia, egg production, disease resistance etc.。Additionally, by injecting external source GH, it is possible to significantly improve Embryo Gallus domesticus weight at embryonic stage, promote the growth of its long bone and cartilaginous tissue, thus improving the market weight of meat breed chicken。Research relative to people and mammiferous growth hormone, the research of chicken growth hormone is started late, but it is as developing rapidly of protein hetero expression, increasing expression system is established and is applied, yeast is as unicellular eukaryote, because having the complete gene expression regulation mechanism of comparison and the processing Modifying Capability to expression product, show incomparable advantage。Wherein Pichia sp. (Pichiapastoris) expression system is development in recent years a kind of eukaryotic expression system rapid, widely used, yet there are no the report that any chicken growth hormone recombiant protein is expressed in Pichia sp.。
Summary of the invention
Goal of the invention: the present invention provides a kind of chicken growth hormone recombiant protein high efficient expression in Pichia sp. and purification process, obtains high-purity and active chicken growth hormone recombiant protein by the method。
Technical scheme: in order to reach foregoing invention purpose, the chicken growth hormone recombiant protein provided by the invention expression in Pichia sp. and purification process, comprise the following steps:
(1) extracting total serum IgE from chicken hypophysis and do reverse transcriptional PCR and obtain its cDNA, with cDNA for template, PCR obtains the genetic fragment of chicken growth hormone, and its base sequence is such as shown in SEQIDNO:1；
(2) step (1) gained genetic fragment is inserted Pichia sp. secretion expression carrier pPIC9K, and add 6 His labels at C end, obtain recombinant expression carrier pPIC9K-cGH；
(3) recombinant expression carrier pPIC9K-cGH electricity converts Pichia pastoris GS115, and obtains transgenic restructuring yeast strains GS115-pPIC9K-cGH by histidine defect screening and G418 screening；
(4) expressed by methanol induction, obtain chicken growth hormone recombiant protein through nickel post, gel column purification。
The gene of described chicken growth hormone (chickengrowthhormone, cGH) is positioned at No. 1 autosomal long-armed end, and total length is about 4kb, is made up of 5 exons and 4 introns。CGH is made up of 191 aminoacid, and molecular weight is about 22KDa, has significantly high homology with the growth hormone of duck, only 4 amino acid whose difference, is 96% with the homology of turkey growth hormone。Chicken growth hormone mRNA sequence (NM_204359) that Genbank has announced。
The base sequence of described chicken growth hormone gene fragment is such as shown in SEQIDNO:1, and the aminoacid sequence of the recombiant protein of its coding is such as shown in SEQIDNO:2。
In the structure of described step (2) recombinant expression carrier, for the ease of subsequent purification recombiant protein, when step (1) gained genetic fragment is inserted Pichia sp. secretion expression carrier pPIC9K, it is necessary to add 6 His labels at C end。Its operation can by means of containing His label pET-30a (+) carrier, particularly as follows: by step (1) gained genetic fragment insert containing His label pET-30a (+) carrier, and with it for template, pcr amplification obtains the genetic fragment of the chicken growth hormone containing His label and inserts Pichia sp. secretion expression carrier pPIC9K, obtains recombinant expression carrier pPIC9K-cGH。Wherein it is possible to first step (1) gained gene to be inserted pMD19-T carrier, called after pMD19-T-cGH, then carry out above-mentioned pcr amplification according to design primer。
Described step (3) is specifically as follows: the pPIC9K-cGH recombinant expression carrier SacI linearisation that will build, and then mixes with Pichia pastoris GS115 competent cell, turns cultivation afterwards through electricity and grows transformant。All transformant sterile deionized water are washed the YPD flat board that lower painting contains variable concentrations G418, cultivates the transformant of screening high copy number。Take to boil-freeze-cooking method extraction transformant genome, be PCR and identify, identify correct height copy transformant called after GS115-pPIC9K-cGH。
It is 1500V, 6ms that described electricity turns condition。
Described step (4) is specifically as follows: the transgenic mono-colony inoculation of restructuring yeast strains GS115-pPIC9K-cGH is cultivated activation in culture medium；Being inoculated in BMGY culture medium by the bacterium solution after activation, cultivate 16-20h to OD600 and reach 2-6,2500 × g, 5min is centrifugal collects thalline, uses BMMY culture medium abduction delivering instead, adds methanol continuous induction, filter out high expressed bacterial strain。
Methanol used by described abduction delivering is the 1% of culture volume。
The described abduction delivering time is 24-96h, and the optimal expression time is 96h。
Nickel post, gel column purification in step (4), particularly as follows: the GS115-pPIC9K-cGH taking 100mL induces supernatant, are dialysed 18h with 8000KDa bag filter in nickel post 2L is in conjunction with liquid, and every 6h changes a not good liquor；By the induction supernatant ni-sepharose purification after dialysis, then adopt gel chromatography method to remove wherein foreign protein, obtain purity and reach the chicken growth hormone recombiant protein of more than 95%。The activity of gained recombiant protein is identified finally by cell activity assays。
Beneficial effect: the present invention realizes the chicken growth hormone recombiant protein high efficient expression in Pichia sp. purification first, thus obtaining highly purified chicken growth hormone recombiant protein；The destination protein produced can direct secretion in culture fluid, convenient separate, and cell can be re-used and carry out induction destination protein, can be applicable to the pattern that continuous fermentation produces, it is greatly improved the generation of destination protein, improves fermentation efficiency, simplify sweat and reduce cost；Avoiding the more produced materials not easily removed such as endotoxin material and thermal source of prokaryotic cell, destination protein purity is higher simultaneously。Namely the inventive method is simple to operate, yield is high, cost is low。
Accompanying drawing explanation
(M is DL2000DNAMaker to Fig. 1: pPIC9K-cGH recombinant vector enzyme action inspection agarose electrophoretic analysis figure；1 is the pPIC9K-cGH recombinant vector of EcoRI, NotI double digestion。The cGH band of an about 600bp and a plasmid band more than 2000bp is produced, it was shown that cGH sequence has been successively inserted into expression vector after double digestion。)
(M is DL2000DNAMaker to Fig. 2: GS115-pPIC9K-cGH Genomic PCR inspection agarose electrophoretic analysis figure；1 is negative control；2-6 is for doing template PCR primer with different transformant genomes。Extraction transformant genome does the PCR band that can obtain being consistent with cGH size and illustrates that pPIC9K-cGH successfully proceeds to GS115。)
(M is PremixedProteinMarker to the SDS-PAGE electrophoretic analysis figure of Fig. 3: GS115-pPIC9K-cGH not isogeneous induction phase culture supernatant；The supernatant that 1 is induction 96h；The supernatant that 2 is induction 72h；The supernatant that 3 is induction 48h；The supernatant that 4 is induction 24h。)
(M is PremixedProteinMarker to Fig. 4: GS115-pPIC9K-cGH induction supernatant ni-sepharose purification SDS-PAGE electrophoretic analysis figure；1 is induction liquid supernatant；2 was post liquid；3,4 is 50mM imidazole wash liquid；5,6 is 100mM imidazole wash liquid；7,8 is 500mM imidazole elution。Result shows, part foreign protein can be washed off when cleaning mixture imidazoles is 50mM, when imidazole concentration improves 100mM, destination protein starts to be washed down, can obtain major part prediction with 500mM imidazoles eluting and be sized to the chicken growth hormone recombiant protein of about 25KDa, but purity is not high。)
Fig. 5: (M is PremixedProteinMarker to chicken growth hormone recombiant protein gel chromatography SDS-PAGE electrophoretic analysis figure；1 recombiant protein obtained for ni-sepharose purification；2,3,4 respectively albumen in gel chromatography difference peak。Single band from 4 it can be seen that can obtain the chicken growth hormone recombiant protein that purity is higher after gel chromatography。)
Fig. 6: LMH cell through chicken growth hormone recombiant protein process after IGF-ImRNA relative to internal reference GAPDH expression block diagram (cGH-be PBS process negative control group；CGH+500 is 500ng/mL chicken growth hormone recombiant protein process group；CGH+1000 is 1000ng/mL chicken growth hormone recombiant protein process group；CGH+1500 is 1500ng/mL chicken growth hormone recombiant protein process group。After chicken growth hormone recombiant protein processes, the IGF-ImRNA expression of LMH cell significantly improves, in extremely notable (p < 0.01) compared with matched group, it was shown that the chicken growth hormone recombiant protein that the present invention obtains has higher biologic activity。
Detailed description of the invention
Embodiment 1: build recombinant expression carrier and the transgenic restructuring yeast strains of chicken growth hormone recombiant protein
1, from chicken hypophysis, obtain the genetic fragment of cGH
From hypophysis, extract total serum IgE and do reverse transcriptional PCR and obtain its cDNA。Chicken growth hormone mRNA sequence (NM_204359) that Genbank has announced designs primer, with cDNA obtained above for template, is PCR and obtains the total length CDS fragment of cGH gene。The genetic fragment of above-mentioned cGH is inserted into pMD19-T carrier, and called after pMD19-T-cGH is, and check order and determine and correctly obtain genes of interest。
2, pPIC9K-cGH recombinant expression carrier is built
Gene order design primer according to chicken growth hormone mature polypeptide, wherein forward primer P1 is with NdeI restriction enzyme site, downstream primer P2 is with XhoI restriction enzyme site: according in pET-30a-cGH recombinant vector, His sequence label design forward primer P3 is with EcoRI restriction enzyme site, and downstream primer P4 is with NotI restriction enzyme site。
P1:5 ' ATcatatgACCTTCCCTGCCATGC 3 '
P2:5 ' ATctcgagGATGGTGCAGTTGCTCTC 3 '
P3:5 ' ATgaattcACCTTCCCTGCCATGC 3 '
P4:5 ' ATgcggccgcTTAGCAGCCGGATCTCA 3 '
In 1, pMD19-T-cGH plasmid is template, carries out pcr amplification with P1, P2 primer。
PCR system:
Reaction condition is: 95 DEG C of denaturation 3min；95 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 40sec, 30 circulations；72 DEG C extend 10min；16 DEG C of end。
PCR primer after NdeI, XhoI double digestion, be connected to pET-30a through NdeI, XhoI double digestion (+) carrier, and convert bacillus coli DH 5 alpha competent cell。The LB flat board that DH5 α after conversion is coated with containing kanamycin screens, cultivate 12h for 37 DEG C, picking list colony inoculation contains the LB fluid medium of kanamycin to 3mL, 12h cultivated by 37 DEG C of shaking tables, after doing bacterium solution PCR qualification, extract plasmid NdeI, XhoI double digestion to identify, and check order, the recombinant vector called after pET-30a-cGH checking order correct。
With above-mentioned pET-30a-cGH recombinant vector for template, carry out pcr amplification with P3, P4 primer。
PCR system:
Reaction condition is: 95 DEG C of denaturation 3min；95 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 40sec, 30 circulations；72 DEG C extend 10min；16 DEG C of end。
PCR primer after EcoRI, NotI double digestion, it is connected to the pPIC9K carrier through EcoRI, NotI double digestion (pPIC9K carrier used flies generation purchased from Sai Mo, and you are scientific and technological, article No.: K1750-01), and converts bacillus coli DH 5 alpha competent cell。The LB flat board that DH5 α after conversion is coated with containing ammonia benzyl screens, cultivate 12h for 37 DEG C, picking list colony inoculation contains the LB fluid medium of ammonia benzyl to 3mL, 12h cultivated by 37 DEG C of shaking tables, after doing bacterium solution PCR qualification, extract plasmid EcoRI, NotI double digestion to identify, and carry out checking order to determine correct carrier construction, recombinant expression carrier called after pPIC9K-cGH (Fig. 1) checking order correct。
3, GS115-pPIC9K-cGH transgenic restructuring yeast strains is built
By the above-mentioned pPIC9K-cGH recombinant expression carrier SacI linearisation built, linearizing for 10ug plasmid and 80 μ L Pichia pastoris GS115 competent cells being mixed, turn painting MD flat board afterwards through 1500V, 6ms electricity, 28 DEG C of incubators are cultivated and are grown transformant in 3-5 days。All transformant sterile deionized water are washed the YPD flat board that lower painting contains variable concentrations G418, and 28 DEG C of incubators are cultivated 3-5 days, the transformant of screening high copy number。Take to boil-freeze-cooking method extraction transformant genome, be PCR and identify, identify correct height copy transformant called after GS115-pPIC9K-cGH (Fig. 2)。
Embodiment 2: the abduction delivering of chicken growth hormone recombiant protein, purification and activity identification
1, chicken growth hormone recombiant protein is expressed with methanol induction
By the transgenic mono-colony inoculation of restructuring yeast strains GS115-pPIC9K-cGH in 3mLYPD culture medium, 18h activation cultivated by 28 DEG C of shaking tables；Being inoculated in BMGY culture medium by above-mentioned bacterium solution according to 1%, 16-20h to OD cultivated by 28 DEG C of shaking tables600Reach 2-6,2500 × g, 5min are centrifugal collects thalline, uses BMMY culture medium abduction delivering instead, every 24h adds methanol to 1%, continuous induction 96h, collects supernatant, SDS-PAGE analyzing proteins (Fig. 3) at 24h, 48h, 72h, 96h respectively, it is incremented by over time, expression effect is more good, and wherein 96h expression effect is best, filters out high expressed bacterial strain therewith。
2, the purification of chicken growth hormone recombiant protein
The GS115-pPIC9K-cGH taking 100mL induces 96h supernatant, dialyses 18h with 8000KDa bag filter in nickel post 2L is in conjunction with liquid, and every 6h changes a not good liquor；By the induction supernatant ni-sepharose purification after dialysis, wherein collected post liquid, cleaning mixture, eluent do SDS-PAGE electrophoretic analysis (Fig. 4)。Containing foreign protein in eluted protein, therefore adopt gel chromatography method to remove wherein impurity, and do SDS-PAGE electrophoretic analysis (Fig. 5)。Operation is in detail: the GS115-pPIC9K-cGH taking 100mL induces 96h culture supernatant, after being loaded in 8000KDa bag filter, it is placed in the dialysis equilibrium liquid (used by affinity chromatography in conjunction with liquid) of 20 times dialysis 18h, every 6h changes a not good liquor, by the induction supernatant 12 after dialysis, 000rpm is centrifuged 30 minutes (4 DEG C), and with the membrane filtration sample of 0.2 μM, 1mL nickel post is balanced with flow velocity for 1mL/min in conjunction with liquid with 10mL (10 column volumes), then by the sample after filtration with flow velocity for 0.5mL/min loading, respectively with containing 50mM imidazoles after end of the sample, 100mM imidazoles wash successively in conjunction with balance liquid (i.e. cleaning mixture) 10 column volumes in remove non-specific binding albumen, finally with 500mM imidazoles in conjunction with balance liquid (i.e. eluent) elution of bound albumen, collect eluting peak, this purge process was collected post liquid simultaneously, cleaning mixture。Then (gel column filler is sephacrylS-100: column length 60cm to adopt gel chromatography, internal diameter 16mm) carry out second time purification destination protein by molecular sieving effect, first with the PBS of pH7.2 with flow velocity one column volume of 0.5mL/min balanced gel post, then sample (the Tot Prot 3mg containing destination protein of first time purification is gone up, volume 5mL), continue by PBS elution samples at a same speed, collecting eluting peak, the destination protein (chicken growth hormone mature polypeptide) purity after twice purification can reach more than 95%。
3, the chicken growth hormone recombiant protein response rate is measured by BCA method
With PBS, the BSA standard protein of 25mg/mL is diluted to the standard substance of 0.5mg/mL, by standard substance by 0,1,2,4,8,12,16,20 μ L are added in the standard sample wells of 96 orifice plates, and PBS supplies 20 μ L, the chicken growth hormone recombiant protein of purification is joined in the testing sample hole of 96 orifice plates, every hole adds 200 μ LBCA working solutions, places 30 minutes, measures absorbance (A by microplate reader for 37 DEG C562nm), with standard substance absorbance drawing standard curve (y=0.9296x-0.0908, R2=0.998, wherein y is protein concentration mg/mL, x is absorbance), calculate sample concentration and be about 0.1mg/mL, finally show that every liter of culture response rate of chicken growth hormone recombiant protein of the present invention reaches more than 10 milligrams。
4, chicken growth hormone recombiant protein activity identification
By LMH cell with 5 × 106Cells/ hole is inoculated in 24 orifice plates, by the culture medium containing serum at 37 DEG C, and 5%CO2Incubator in cultivate；After adhere-wall culture 24h, change serum-free medium and continue to cultivate 8h；With cGH recombiant protein process cell, matched group PBS process, 37 DEG C, 5%CO2After processing 12h, extract cell total rna；The expression of IGF-ImRNA is affected by fluorescence quantifying PCR method detection restructuring cGH albumen。Process after 12h through chicken growth hormone recombiant protein, the expression of LMH cell IGF-ImRNA significantly increases, showing that the chicken growth hormone recombiant protein that the present invention obtains can urge the LMH cell IGF-ImRNA rise expressed, it has biologic activity (Fig. 6)。Provide the foundation for studying the structure of chicken growth hormone, function and production application further。
Claims (6)
1. expression in Pichia sp. of a chicken growth hormone recombiant protein and purification process, it is characterised in that the method comprises the following steps:
(1) extracting total serum IgE from chicken hypophysis and do reverse transcriptional PCR and obtain its cDNA, with cDNA for template, PCR obtains the genetic fragment of chicken growth hormone；
(2) step (1) gained genetic fragment is inserted Pichia sp. secretion expression carrier pPIC9K, and add 6 His labels at C end, obtain recombinant expression carrier pPIC9K-cGH；
(3) recombinant expression carrier pPIC9K-cGH electricity converts Pichia pastoris GS115, and obtains transgenic restructuring yeast strains GS115-pPIC9K-cGH by histidine defect screening and G418 screening；
(4) expressed by methanol induction, obtain chicken growth hormone recombiant protein through nickel post and gel column purification。
2. the chicken growth hormone recombiant protein according to claim 1 expression in Pichia sp. and purification process, it is characterized in that step (2) is as follows: by step (1) gained genetic fragment insert containing His label pET-30a (+) carrier, and with it for template, pcr amplification obtains the genetic fragment of the chicken growth hormone containing His label and inserts Pichia sp. secretion expression carrier pPIC9K, obtains recombinant expression carrier pPIC9K-cGH。
3. the chicken growth hormone recombiant protein according to claim 1 expression in Pichia sp. and purification process, it is characterised in that: it is 1500V, 6ms that step (3) electricity turns condition。
4. the chicken growth hormone recombiant protein according to claim 1 expression in Pichia sp. and purification process, it is characterised in that: step (4) induction methanol used is the 1% of culture volume。
5. the chicken growth hormone recombiant protein according to claim 1 expression in Pichia sp. and purification process, it is characterised in that: step (4) the abduction delivering time is 96h。
6. the chicken growth hormone recombiant protein according to claim 1 expression in Pichia sp. and purification process, it is characterized in that the nickel post of step (4), gel column purification particularly as follows: the GS115-pPIC9K-cGH taking 100mL induces supernatant, dialysing in nickel post 2L is in conjunction with liquid 18h with 8000KDa bag filter, every 6h changes a not good liquor；Induction supernatant after dialysis is first used ni-sepharose purification, then adopts gel chromatography method to remove wherein foreign protein。
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