CN102876708A - Method for heterologously expressing active membrane proteins by using Pichia pastoris expression system - Google Patents
Method for heterologously expressing active membrane proteins by using Pichia pastoris expression system Download PDFInfo
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- CN102876708A CN102876708A CN2012104144864A CN201210414486A CN102876708A CN 102876708 A CN102876708 A CN 102876708A CN 2012104144864 A CN2012104144864 A CN 2012104144864A CN 201210414486 A CN201210414486 A CN 201210414486A CN 102876708 A CN102876708 A CN 102876708A
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Abstract
The invention discloses a method for heterologously expressing active membrane proteins by using a Pichia pastoris expression system. The invention relates to a method for expressing a myosin cross-reactive antigen (MCRA) of Bifidobacterium BB-12 in a recombinant mode and an expression host used in the method. The expression host is a PichiaPinkTM expression system transformed from pPink alpha-HC-MCRA plasmid. The invention also relates to application of an MCRA of Bifidobacterium BB-12 or an expression host in preparing conjugated linoleic acid.
Description
[technical field]
The present invention relates to genetically engineered and zymotechnic field, more specifically, the present invention relates to a kind of activity expression, enzymatic function and scientific research purposes thereof of membranin.
[background technology]
Pichia pastoris phaff (Pichia pastoris) is a kind of yeast kind of energy highly effective expressing recombinant protein, compare with other expression system, pichia yeast expression system has following advantage: 1) distinctive strong AOX(alcohol oxidase gene) promotor, methyl alcohol can strictly be regulated and control the expression of foreign gene, and methyl alcohol induces the IPTG of use cheap than the general escherichia coli expression that is used for; 2) expression level is high, can be at intracellular expression, but secretion type expression again; 3) zymotechnique is ripe, easily amplifies; 4) the cultivation cost is low, and product is easily separated; 5) foreign protein genes inheritance stability.General foreign protein genes is incorporated on the pichia spp karyomit(e), copies with chromosome duplication, is difficult for losing; 6) as eukaryotic expression system, pichia spp has Eukaryotic subcellular structure, has the posttranslational modification machining functions such as glycosylation, fatty acidylate, protein phosphorylation.
Pichia yeast expression system PichiaPink
TMCompared to common pichia pastoris phaff system many below several advantages: utilize VITAMIN B4 auxotroph (knocking out the ADE2 gene) to screen, avoided the loaded down with trivial details of resistance screening; Knock out proteolytic enzyme pep4 and/or prb1 gene, reduced the degraded of target protein; Having different carriers for different albumen selects, low copy carrier, high copy vector are arranged, with a high copy vector with α-mating signal signal peptide gene, wherein the high copy vector pPink α-HC with signal peptide is particularly suitable for soluble protein is secreted to born of the same parents, the protein purification that is conducive to the later stage, also can guarantee in addition the location of membranin, to guarantee its active performance, when this has been avoided that also gene is recombinant expressed in intestinal bacteria, because of the situation of protein localization failure inactivation.
Milk-acid bacteria with product conjugated linolic acid (CLA) has obtained wide coverage, as: the efficient that lactobacillus reuteri, plant lactobacillus, Lactobacterium acidophilum, bifidobacterium breve and animal bifidobacteria etc. produce CLA is very high.Animal bifidobacteria BB-12 is a kind of bacterium wherein, this bacterium be the known bacterium of prior art (for example referring to Liu Yong, Zhang Yong, Bao Yan, the surface property of a peaceful .4 probiotics and suppress pathogenic bacterium effect research. the Chinese food journal .2010. second phase 2; Wang Jicheng, Guo Zhuan, Yan Liya, Liu Xiaoming, Chen Wei, a peace. probiotic bacterium Lactobacillus casei Zhang and commercial probiotic bacterium are to the comparison of gastrointestinal transit tolerance and fermentation character. the 5th phase of Chinese food journal .2009.), can obtain by commercial the purchase.This bacterium can be converted into CLA with 50% substrate linolic acid (LA), and has bioactively along 9 among the CLA that produces, and the content of anti-11-CLA surpasses 50%.
Although it is existing a lot of that milk-acid bacteria produces the domestic and foreign literature report of conjugated linolic acid, but milk-acid bacteria produces the mechanism of CLA and is still not clear, particularly the enzyme in catalyzed reaction is likely in the situation of membranin, relevant progress is very slow, it is almost nil at present to utilize related microorganisms to carry out the progress of scale operation, and nutritionists are enlarging the output of conjugated linolic acid by every means for use in medical treatment or protective foods.
[summary of the invention]
The object of the invention is according to existing membranin activity expression than hard problem, provides a kind of method of activity expression of restructuring to the myosin cross reaction (MCRA) that comes from animal bifidobacteria BB-12, and described method is at PichiaPink
TMExpress the membranin MCRA of animal bifidobacteria BB-12 in the expression system.When the method for myosin cross reaction (MCRA) antigen of recombinant expressed bifidus bacillus BB-12: take animal bifidobacteria BB-12 genome as template, with 5 '-CCGGATATCATGGACACTAGGGCGCCGAAAGTCG-3 ', 5 '-CGGGGTACCTCAGTGATGGTGATGGTGATGTTTCGCCGAATCATTCTCCCCCG-3 ' is primer, obtains the encoding gene of MCRA albumen by the method for High fidelity PCR amplification.Behind the encoding gene that obtains MCRA albumen, encoding gene is connected with the carrier pPink α-HC with signal peptide obtains pPink α-HC-MCRA plasmid, then use pPink α-HC-MCRA Plasmid Transformation PichiaPink
TM, transform successful PichiaPink
TMBacterium is expressed the membranin MCRA of animal bifidobacteria BB-12.
Another object of the present invention also relates to the host of myosin cross reaction (MCRA) antigen of a kind of recombinant expressed bifidus bacillus BB-12, and this host is the PichiaPink of pPink α-HC-MCRA Plasmid Transformation
TMBacterium, pPink α wherein-HC-MCRA plasmid are that the MCRA encoding gene with bifidus bacillus BB-12 is connected with the carrier pPink α-HC with signal peptide and obtains.
Another object of the present invention is to provide the application of above-mentioned membranin or activity expression system, can be with bifidus bacillus BB-12 myosin cross reaction (MCRA) antigen that obtains or host for the preparation of conjugated linolic acid.
Above-mentioned purpose of the present invention is to be achieved by the following technical programs:
The present invention clones from animal bifidobacteria BB-12 and obtains myosin cross-reacting antigen (MCRA) gene, and realize its method for activity expression be: with the membranin MCRA of animal bifidobacteria BB-12, with carrier pPink α-HC with signal peptide at PichiaPink
TMExpress in the expression system, correctly be positioned cytolemma, the activated recombinant protein of tool.
Above-mentioned preparation method's step is as follows:
(1) take animal bifidobacteria BB-12 genome as template, with 5 '-CCGGATATCATGGACACTAGGGCGCCGAAAGTCG-3 ', 5 '-CGGGGTACCTCAGTGATGGTGATGGTGATGTTTCGCCGAATCATTCTCCCCCG-3 ' is primer, and the method that increases by High fidelity PCR obtains the MCRA gene.The PCR program is: 95 ℃ of 30s, 55 ℃ of 30s, 68 ℃ of 2.5min, 30 circulations.PCR reaction system: 5 μ L dNTPs (2mM), 5 μ L10 * KOD plus Buffer, 1 μ L KOD plus, 2.5 μ L MgSO
4(25mM), each 1 μ L of upstream and downstream primer, template 2 μ L;
(2) the PCR product that obtains of amplification is behind EcoR V and 37 ℃ of digestion of Kpn I 3h, with be connected with pPink α after Kpn I enzyme is connected-HC plasmid through Stu I, be converted into E.coli TOP10 competent cell and evenly coat LBA flat board (the LB agar plate that contains 100 μ g/mL penbritins), after 37 ℃ of incubated overnight, select mono-clonal;
(3) with correct recombinant plasmid transformed to PichiaPink
TMCompetent cell, it is dull and stereotyped to coat the PAD selectivity that does not contain VITAMIN B4, selects mono-clonal;
(4) with the methanol induction recombination microzyme, express and the location situation by method validation target proteins such as SDS-PAGE and Western Blot, the lipid acid composition of the thalline after inducing and substratum is identified to measure the activity of target protein by GC-MS.
The MCRA albumen of animal bifidobacteria BB-12 of the present invention successfully is positioned to cytolemma.
The MCRA albumen of bifidus bacillus BB-12 of the present invention can be at pichia yeast expression system PichiaPink
TMMiddle expression is expressed the MCRA albumen that obtains and is had activity, can change hydroxylation derivative into by the catalysis linolic acid.
Compared with prior art, the present invention has following beneficial effect:
Myosin cross reaction (MCRA) albumen of bifidus bacillus BB-12 of the present invention had both been realized activity expression in pichia spp, realized again being positioned to cytolemma.
[description of drawings]
Fig. 1 is MCRA gene PCR amplification figure.1 is the PCR product, 2 negative contrasts;
Fig. 2 is pPink α-HC-MCRA plasmid proof diagram.1 for making the pcr amplification of template with recombinant plasmid, 2 for to make positive control with genome, and 3 for to make negative control with empty pPink α-HC plasmid;
Fig. 3 is recombinant yeast pichia pastoris PichiaPink
TMPPink α-HC-MCRA recon proof diagram.1 makes negative control for original bacterium genome, and 2 for the integron genes group is template, and 3 for to make positive control with corresponding plasmid
Fig. 4 is the SDS-PAGE(A of restructuring PichiaPinkTM bacterium) and Western Blot(B) figure.1 is Pichia yeast fermented liquid supernatant sample, and 2 is Pichia yeast thalline sample;
Fig. 5 is the time restructuring PichiaPink that do not add substrate
TMThe thalline of bacterium and fermented liquid supernatant lipid acid GC-MS detect as a result figure of GC.A, B are respectively thalline and the fermented liquid supernatant fatyy acids result of contrast bacterium, and C, D are respectively thalline and the fermented liquid supernatant fatyy acids result of recombinant bacterium;
Fig. 6 is restructuring PichiaPink when adding substrate
TMThe thalline of bacterium and fermented liquid supernatant lipid acid GC-MS detect as a result figure of GC.A, B are respectively thalline and the fermented liquid supernatant fatyy acids result of contrast bacterium, and C, D are respectively thalline and the fermented liquid supernatant fatyy acids result of recombinant bacterium;
Fig. 7 is the actual and theoretical fragment of the MS of product.
[embodiment]
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of restriction to the present invention.
Change the PichiaPink of the MCRA gene of animal bifidobacteria BB-12 over to
TMThe foundation of recombinant bacterium.
(1) test method: take the genome of animal bifidobacteria BB-12 as template, according to MCRA sequence (GenBank:ADC85468) the design primer of animal bifidobacteria BB-12, carry out pcr amplification MCRA gene with the KOD plus enzyme of high-fidelity.With 5 '-CCGGATATCATGGACACTAGGGCGCCGAAAGTCG-3 ', 5 '-CGGGGTACCTCAGTGATGGTGATGGTGATGTTTCGCCGAATCATTCTCCCCCG-3 ' is primer, and the method that increases by High fidelity PCR obtains the MCRA gene.The PCR program is: 95 ℃ of 30s, 55 ℃ of 30s, 68 ℃ of 2.5min, 30 circulations.PCR reaction system: 5 μ L dNTPs (2mM), 5 μ L10 * KOD plus Buffer, 1 μ L KOD plus, 2.5 μ L MgSO
4(25mM), each 1 μ L of upstream and downstream primer, template 2 μ L.The PCR product that obtains of amplification and is connected with suitable ratio with pPink α after Kpn I enzyme is connected-HC plasmid through Stu I behind EcoR V and Kpn I37 ℃ of digestion 3h.Connect product Transformed E .coli TOP10 competent cell, and evenly coat LBA flat board (the LB agar plate that contains 100 μ g/mL penbritins), 37 ℃ of incubated overnight are screened.Select transformant, carry out PCR checking and sequence verification, obtain correct recombinant plasmid pPink α-HC-MCRA.The electricity consumption conversion instrument with the linearizing pPink α after Spe I enzyme is cut-HC-MCRA Plasmid Transformation to PichiaPink
TMBacterium competence cell is by the positive bacterial strain that transforms of PAD selectivity plate screening.The picking transformant take the genome of recombinant yeast pichia pastoris bacterium as template, carries out the PCR checking, filters out the correct recombinant bacterium of homologous recombination and changes the contrast bacterium of empty plasmid over to.Preserve simultaneously the correct recombinant bacterium of homologous recombination that filters out, as the bacterial classification of enlarged culturing.
(2) result:
The length of the length of PCR cloned sequence and MCRA gene itself is complementary, represent successfully to clone MCRA gene (see figure 1), the MCRA gene successfully is connected to (see figure 2) on pPink α-HC plasmid, plasmid checks order by the biological company limited of Shanghai Sani, and the result shows MCRA gene entirely true (GenBank:ADC85468).The MCRA gene is connected resulting pPink α-HC-MCRA plasmid and successfully is integrated in the genome of PichiaPinkTM bacterium with pPink α-HC plasmid, obtained simultaneously integrating the PichiaPinkTM contrast bacterium (see figure 3) of empty plasmid pPink α-HC, wherein pPink α-HC plasmid and PichiaPink
TMExpression system is available from Invitrogen.
The MCRA gene of 1 animal bifidobacteria BB-12 is at PichiaPink
TMThat expresses in the recombinant bacterium determines.
1.1 experimental technique: recombinant bacterium and contrast bacterium carry out SDS-PAGE and Wester Blot check to thalline and substratum albumen partly behind methanol induction.
(1) solution preparation:
BMGY substratum: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer pH6.0,1.34%YNB, 0.0004% vitamin H, 1% glycerine; Glycerine among the BMGY substituted with 1% methyl alcohol namely get the BMMY substratum;
(2) recombinant bacterium and contrast bacterium were cultivated 2 days under 28 ℃, 250rpm condition in the BMGY substratum, the centrifugal 5min of rear 1500g, remove the BMGY substratum, with the resuspended thalline of BMMY substratum, in 28 ℃, 250rpm inducing culture after 2 days, get respectively that fermented liquid, thalline carry out SDS-PAGE and Western Blot analyzes, resolving gel concentration is 12%.
(3) SDS-PAGE and Western Blot
10 μ g samples are carried out electrophoresis at two 12% polyacrylamide gel, and 80V concentrates 25min, and 150V separates 60 ~ 80min again.Wherein one with coomassie brilliant blue staining and acetic acid-ethanolic soln decolouring, and analyzing proteins distributes and concentration in gel imaging system.Another piece glue is gone to pvdf membrane, after successively in conjunction with primary antibodie (antibody of anti-His) and two anti-(Horseradish peroxidase (HRP)-conjugated anti-mouse IgG), process with enhancement type fluorescent reagent box at last and develop.
1.2 experimental result: SDS-PAGE and Western Blot analyze and show that recombination obtains expressing in the PichiaPinkTM recombinant bacteriums, and are positioned cytolemma, and molecular weight is that 82kDa(sees Fig. 4).
2, the MCRA albumen of animal bifidobacteria BB-12 is at PichiaPink
TMActive determining in the recombinant bacterium.
(1) test method: after recombinant bacterium and contrast bacterium are induced 1 day as stated above, one group is added the substrate linolic acid, one group is not added linolic acid, after continuing to induce 1 day, receive the bacterium freeze-drying, the thalline lyophilized powder is pressed literature method (Sakuradani E, Shimizu S.Gene cloning and functional analysis of a second Δ 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus.Biosci Biotech Bioch 2003,67:704 – 711) carries out lipids extraction.Add the hydrochloric acid-methanol solution of mark in the nondecylic acid and 10% in the 200mg thalline lyophilized powder, 60 ℃, hatch 3h.Rear adding normal hexane and saturated NaCl, the vibration mixing, centrifugal rear supernatant is in clean bottle; Repeat to extract once; N
2Dry up rear with the additional esterification of diazomethane, N
2Dry up the rear normal hexane Hui Rong that uses, be used for GC-MS and detect analysis.Nutrient solution also carries out lipids extraction and esterification processing by same procedure after the 1mL fermentation.The GC-MS condition: Finnigan Trance GC/Finnigan Trance MS(U.S. Thermo), chromatographic column: Agilent DB-WAX(30m * 0.250mm id * 0.25 μ m, U.S.'s Agilent).The GC heating schedule: 180 ℃ keep 0.5min, and the temperature rise rate with 5 ℃/min rises to 230 ℃ afterwards, keep 13min.Carrier flow He:8mL/min, carrier gas flux He:8mL/min, the vaporizer temperature: 250 ℃, sample size: 10 μ L, splitting ratio: 62:1.Mass spectrometric detection condition: ionization mode: EI, ionization voltage: 70eV; Transmitter current 200 μ A; Temperature is 250 ℃ on the post; 230 ℃ of ion chamber's temperature; Detector voltage 350V.
(2) test-results: only when adding the substrate linolic acid, detect corresponding product in the thalline of recombinant bacterium, product is 10-hydroxyl-suitable-12-octadecenoic acid, and namely 10-HOE(sees Fig. 5, Fig. 6, Fig. 7).The result shows that the MCRA albumen of recombinant bacterium expression can change into linolic acid the ability of conjugated linolic acid 10-hydroxyl-suitable-12-octadecenoic acid, and the contrast bacterium does not possess this ability.
Claims (7)
1. the method for myosin cross reaction (MCRA) antigen of a recombinant expressed bifidus bacillus BB-12 is characterized in that at PichiaPink
TMExpress the membranin MCRA of animal bifidobacteria BB-12 in the expression system.
2. the method for myosin cross reaction (MCRA) antigen of recombinant expressed bifidus bacillus BB-12 according to claim 1, it is characterized in that the method also comprises: take animal bifidobacteria BB-12 genome as template, with 5 '-CCGGATATCATGGACACTAGGGCGCCGAAAGTCG-3 ', 5 '-CGGGGTACCTCAGTGATGGTGATGGTGATGTTTCGCCGAATCATTCTCCCCCG-3 ' is primer, obtains the encoding gene of MCRA albumen by the method for High fidelity PCR amplification.
3. the method for myosin cross reaction (MCRA) antigen of recombinant expressed bifidus bacillus BB-12 according to claim 2, it is characterized in that the method also comprises: behind the encoding gene that obtains MCRA albumen, encoding gene is connected with the carrier pPink α-HC with signal peptide obtains pPink α-HC-MCRA plasmid, then use pPink α-HC-MCRA Plasmid Transformation PichiaPink
TM, transform successful PichiaPink
TMBacterium is expressed the membranin MCRA of animal bifidobacteria BB-12.
4. the plasmid of myosin cross reaction (MCRA) antigen of a recombinant expressed bifidus bacillus BB-12 is characterized in that this plasmid is pPink α-HC-MCRA.
5. the host of myosin cross reaction (MCRA) antigen of a recombinant expressed bifidus bacillus BB-12 is characterized in that, this host is the PichiaPink of pPink α-HC-MCRA Plasmid Transformation
TMBacterium, pPink α wherein-HC-MCRA plasmid are that the MCRA encoding gene with bifidus bacillus BB-12 is connected with the carrier pPink α-HC with signal peptide and obtains.
6. bifidus bacillus BB-12 myosin cross reaction (MCRA) albumen is for the preparation of the purposes of conjugated linolic acid.
7. plasmid claimed in claim 4 or host claimed in claim 5 are for the preparation of the purposes of conjugated linolic acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106566840A (en) * | 2016-11-01 | 2017-04-19 | 中国科学院华南植物园 | Improved pichia pastoris protein expression vector and construction method thereof |
| CN107779458A (en) * | 2016-08-29 | 2018-03-09 | 中国科学院上海巴斯德研究所 | A kind of virus-like particle of rabies viruses of yeast cell to express and preparation method thereof |
| CN108315344A (en) * | 2018-02-14 | 2018-07-24 | 武汉博沃生物科技有限公司 | VZV glycoprotein E genes expression vector and its restructuring yeast strains and application |
| CN113061542A (en) * | 2020-03-23 | 2021-07-02 | 江南大学 | Pichia pastoris engineering bacterium capable of producing conjugated linoleic acid and application thereof |
-
2012
- 2012-10-26 CN CN2012104144864A patent/CN102876708A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| EVA ROSBERG-CODY 等: "Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection", 《BMC BIOCHEMISTRY》 * |
| GARRIGUES,C.等: "Myosin-crossreactive antigen [Bifidobacterium animalis subsp. lactis BB-12", 《NCBI DATABASE》 * |
| INVITROGEN: "《PichiaPink™ Expression System，User Manual》", 6 August 2010 * |
| M. COAKLEY 等: "Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779458A (en) * | 2016-08-29 | 2018-03-09 | 中国科学院上海巴斯德研究所 | A kind of virus-like particle of rabies viruses of yeast cell to express and preparation method thereof |
| CN107779458B (en) * | 2016-08-29 | 2023-06-20 | 中国科学院上海巴斯德研究所 | Virus-like particle of rabies virus expressed by yeast cells and preparation method thereof |
| CN106566840A (en) * | 2016-11-01 | 2017-04-19 | 中国科学院华南植物园 | Improved pichia pastoris protein expression vector and construction method thereof |
| CN108315344A (en) * | 2018-02-14 | 2018-07-24 | 武汉博沃生物科技有限公司 | VZV glycoprotein E genes expression vector and its restructuring yeast strains and application |
| CN113061542A (en) * | 2020-03-23 | 2021-07-02 | 江南大学 | Pichia pastoris engineering bacterium capable of producing conjugated linoleic acid and application thereof |
| CN113061542B (en) * | 2020-03-23 | 2023-06-16 | 江南大学 | Pichia pastoris engineering bacteria capable of producing conjugated linoleic acid and application thereof |
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