CN104046642A - Fermentative production method of dimerized fusion protein - Google Patents
Fermentative production method of dimerized fusion protein Download PDFInfo
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Abstract
The invention discloses construction and fermentation of a dimerized thrombopoietin (TPO) mimic peptide TMP diad-human serum albumin fusion protein. The fusion protein contains an HAS and two TPO mimic peptides which are connected through a linkage oligopeptide and a dimerization domain. A fermentation strategy of the dimerized thrombopoietin mimic peptide TMP diad-human serum albumin fusion protein provided by the invention in pichia pastoris can significantly enhance the expression efficiency of the target protein.
Description
Technical field
The invention belongs to genetic engineering pharmaceutical field, particularly relate to a kind of method of expressing and producing Dimerized fusion rotein in pichia spp in induction type mode.
Background of invention
Pichia spp (Pichia pastoris) is the exogenous protein expression Host Strains being most widely used, and it is the one in methyl alcohol nutritional type yeast, can in the substratum taking methyl alcohol as sole carbon source, grow (Xiong Xianghua, 2006).Pichia spp is one of the most ripe so far exogenous protein expression system, and oneself has hundreds of foreign protein genes in pichia spp, to obtain expression at present.The expression of foreign gene in pichia spp need to have several primary conditions: the one, need to there be efficient promotor and terminator; The 2nd, there is homologous sequence so that foreign gene can be incorporated in pichia spp genome effectively; The 3rd, there is high-throughout screening method.In addition, affect culture condition, the Preference of codon and the tertiary structure of protein etc. in addition that foreign gene is expressed in pichia spp.
The inducible promoter being most widely used is at present pichia spp alcohol oxidase (Alcohol Oxidase, AOX) promotor, in pichia spp, there are two different gene codified alcohol oxidases: be respectively AOX1 and AOX2, between the two, have 97% amino acid sequence homology.In Pichia pastoris, the expression of Gene A OX1 plays a major role to alcohol oxidase activity.The first step of methyl alcohol metabolism is methanol oxidation, generates formaldehyde and hydroperoxide.For avoiding cell hydroperoxide poisoning, methyl alcohol metabolism is carried out in peroxysome.The expression of AOX1 gene is subject to strict regulation and control, and be subject to methanol induction and can reach very high level, be typically when Pichia pastoris is when growing in the substratum of methyl alcohol, AOX1 expression product can account for the more than 30% of cell total soluble protein, and that AOX2 expresses is very low.
Pichia spp has plurality of advantages as exogenous protein expression system, and it belongs to lower eukaryotes, has eukaryote and procaryotic feature concurrently.Pichia spp is unicellular organism; the research of genetic background is relatively thorough; oneself is comparatively clear for its physio-biochemical characteristics; genetically engineered for it operates with respect to plant or zooblast; easier and quick, meanwhile, it has possessed again the unexistent characteristic of prokaryotic organism as eukaryote; glairy translation post-treatment and modification, comprise formation, glycosylation and the acyl etc. of disulfide linkage.For cytotoxic external source recombinant protein, it can be positioned in the peroxysome of pichia spp, both can ensure that host cell is not injured by toxic protein, can prevent again the degraded of proteolytic enzyme to external source recombinant protein.And for the albumen of extracellular expression, because the secretory protein of pichia spp itself is little, therefore, be beneficial to separation and the purifying of external source recombinant protein.Pichi strain and meta-bolites thereof all do not have toxicity for any Mammals, and it can carry out high density fermentation, cultivate with low costly, simple to operate, and the utmost point is suitable for industrialized large scale fermentation and produces.
Adopt pichia spp now comparatively ripe as the technology of expression vector fermentative production recombinant protein, conventionally the fermentation strategies " Pichia Fermentation Process Guidelines " that has used invitrogen company in fermenting process, idiographic flow is summarized as follows:
1 increases the bacterium stage: minimal medium, and glycerine is as carbon source, and Feeding ammonia water supplements nitrogenous source, and adjusts pH to 6.0 left and right.
The 2 abduction delivering stages: inorganic salt are cultivated, and stream adds methyl alcohol as carbon source, and Feeding ammonia water is as nitrogenous source, and adjust pH to 6.0 left and right.
Extensive gradually along with the application of this expression system, increasing different types of fusion rotein is all attempted adopting Bichi yeast system to carry out abduction delivering, but during the fermentation, because different exogenous origin gene integrators causes that the change of thalline character causes recombinant characteristic to have larger difference, therefore adopts the fermentation strategies of traditional common often to can not get desirable ferment effect after entering Host Strains karyomit(e).
The invention discloses structure and the fermentation of Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins.Described fusion rotein comprises 1 HSA and connects by being connected small peptide and Dimerized structural domain with 2 TPO simulating peptide TMP, described fusion rotein is transformed into pichia spp Host Strains, screening has obtained the pichia spp Host Strains that is integrated with antigen-4 fusion protein gene, and this engineering bacteria effective expression in the time of shaking flask has gone out target protein.But in the time that we carry out upper tank fermentation according to the fermentation strategies of invitrogen company " Pichia Fermentation Process Guidelines " by the pichia spp Host Strains that is integrated with antigen-4 fusion protein gene, find that Host Strains cannot run up to the biomass of expectation, also cannot realize effective abduction delivering of target protein.For the problems referred to above, the invention provides the fermentation strategies of a kind of Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins in pichia spp, this fermentation strategies not only suitable engineering bacteria growth and also can significantly improve the productive rate of engineering bacteria abduction delivering target protein.
Summary of the invention
The object of the present invention is to provide a kind of method of expressing Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins in recombinant yeast pichia pastoris.
Another object of the present invention is to provide a kind of method of high-efficiency fermenting restructuring fermentation pichia spp Restruction albumen.
The DNA sequence dna that the invention provides a Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins of coding, its nucleotide sequence is as shown in SeqID No.1.
Dimerized thrombopoietin simulating peptide IMP diad-human serum albumin fusion proteins of the present invention, its aminoacid sequence is as shown in SeqID No.2.
The DNA sequence dna of the Dimerized thrombopoietin simulating peptide of coding of the present invention TMP diad-human serum albumin fusion proteins, this sequence be reconstituted in pPink α-HC or other can abduction delivering and secrete in the plasmid vector of recombinant protein outside born of the same parents after, this recombinant plasmid vector is integrated in yeast host cell karyomit(e).
Yeast host cell of the present invention is Pichi strain PichiaPink
tMexpression Strains or other can expression alien gene yeast strain.
The present invention also provides the method for utilizing the high efficient expression of described pichia spp PichiaPinkTM Expression Strains and producing the Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins of coding, comprising with the next stage:
A: preparation seed liquor: picking is kept at the yeast list bacterium colony that is integrated with SeqID No.1DNA sequence of-20 DEG C, be inoculated in 10mL YPD liquid nutrient medium, 30 DEG C of shaking culture are spent the night, transfer in 100mL YPD liquid nutrient medium with the inoculum size of 10%v/v, 30 DEG C of shaking culture 24 hours, transfer in 1LBMGY substratum with the inoculum size of 10%v/v again, 30 DEG C of shaking culture 16-24 hour, while reaching OD600=5-20 to the yeast amount in fermented liquid, stop fermentation, above-mentioned fermented liquid is as the seed liquor of ferment tank.
B: yeast culture: held 7L basis salt fermention medium in 20L fermentor tank, contained 40g100% glycerine, 26.7ml85%v/v phosphoric acid in the salt fermention medium of every 1L basis, 0.93g calcium sulfate, 18.2g potassium sulfate, 14.9g bitter salt, 4.13g potassium hydroxide, cultivates 20-28 hour.
C: stream adds carbon source: by peristaltic pump flow feeding liquid, the aqueous glycerin solution that feed supplement liquid is 50%v/v, wherein contains 12ml PTM1/L, and stream dosage is 10ml/hr/L, 2-8 hour are cultivated in 30 DEG C of aeration-agitations, reach 180-220g/L to fermented liquid thalline weight in wet base;
D: abduction delivering: pulsed stream adds 100% methyl alcohol in three stages, contains 12ml/L PTM1 in described methyl alcohol, ensure that the dissolved oxygen amount in fermented liquid is greater than 20%, 26-28 DEG C of aeration-agitation cultivation 60-96 hour all the time:
First stage: 2ml/hr/L stream of pulses adds 100% methyl alcohol 8 hours, in described methyl alcohol, contain 12ml PTM1/L, this stage stream adds the citric acid of 0.5M and adjusts fermented liquid pH at 5.90-6.00.
Subordinate phase: 4ml/hr/L stream of pulses adds 100% methyl alcohol 4 hours, contains 12ml PTM1/L in described methyl alcohol, this stage stream adds 50%v/v ammoniacal liquor and adjusts fermented liquid pH at 5.90-6.00.
Phase III: 8ml/hr/L stream of pulses adds methyl alcohol 48-72 hour to fermentation ends, contains 12ml PTM1/L in described methyl alcohol, this stage stream adds 50%v/v ammoniacal liquor and adjusts fermented liquid pH at 5.90-6.00.
Brief description of the drawings
Fig. 1. the gene fragment of Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins builds route map
Fig. 2 .1 pichia spp Fermentation Process of Parameter (experimental group)
Fig. 3 .1 pichia spp Fermentation Process of Parameter (control group)
Fig. 4 .1 pichia spp fermentation results (experimental group): No. 1 swimming lane is standard protein molecular weight; No. 2 swimming lanes are HSA albumen; No. 3 swimming lanes are final tunnings
Fig. 5 .1 pichia spp fermentation results (control group): No. 1 swimming lane is standard protein molecular weight; No. 2 swimming lanes are HSA albumen; No. 3 swimming lanes are final tunnings
Specific embodiment
Structure and the screening of the Dimerized thrombopoietin simulating peptide of embodiment mono-TMP diad-human serum albumin fusion proteins expression vector
One. the structure that comprises TMP-L1-TMP-L2-H-fip-HSA carrier
With Yeast expression carrier pPink α-HC (invitrogen, USA) be template, utilize primer P12 (cccgctttttggatgatt), P13 (gacttccggagccgccaccggccctagcagccagccactgccgcagtgtggggcct tcgat ccttttctcgagagatacc), obtain α-factor-TMP-L1 by pcr amplification, gene order is as shown in Seq ID No:3, and aminoacid sequence is as shown in Seq ID No:4.
With plasmid pcDNA3.1-fip-HSA, (plasmid pcDNA3.1 is purchased from Invitrogen, fip-HSA is synthetic by the full gene of the precious biotech firm in Dalian) be template, adopt primer 1 (gaaaccactctagagaagtgctgtg), (cggggtacctcagtggtggtggtggtggtgtaagcctaaggcagcttgacttgcag c) for primer 2, obtain HSA (post)-His by pcr amplification, gene order is as shown in Seq ID No:5, and aminoacid sequence is as shown in Seq ID No:6.
In above-mentioned pcr amplification reaction, template DNA consumption is 250ng, and primer is 100pM, and polymerase is EasyA Polymerase (Stratagene, USA), adopts 50 μ l systems, 30 circulating reactions (94 DEG C of denaturation 3min; 94 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, 72 DEG C are extended 2min).
The synthetic TMP-L2-H-fip-HSA (pre) of full gene, gene order is as shown in Seq ID No:7, and aminoacid sequence is as shown in Seq ID No:8.
Under similarity condition, utilize pPink α-HC carrier (invitrogen, USA) of SacI and KpnI double digestion 5 μ g, and reclaim acquisition pPink α-HC carrier segment (6902bp).Get 5 μ g α-factor-TMP-L1, process and reclaim respectively with restriction enzyme BspEI and SacI, get 5 μ gHSA (post) fragments, use respectively restriction enzyme KpnI, XbaI processes and reclaims (Fig. 1).
Three fragments connections that restriction enzyme was processed obtain human serum albumin-thrombopoietin fusion gene fragment, connect in Yeast expression carrier pPink α-HC above by molecular cloning means, finally construct pichia spp recombinant plasmid pPink α-TMP-L1-TMP-L2-H-fip-HSA (Fig. 1).Method is as follows:
pPinkα-HC(Sac I/Kpn I) 1.5μl
TMP-L2-H-fip-HSA(pre)(BspEI/XbaI) 6μl
α-factor-TMP-L1(BspEI/SacI) 7μl
HSA(Post)-His(KpnI/XbaI) 7μl
10×T4ligase buffer 2.5μl
T4DNAligase 1μl
16 DEG C, spend the night after ligation and carry out conversion reaction: get and connect product 4 μ l and 50 μ l TOP10 competent escherichia coli cell (invitrogen, USA) after mixing, pack in 0.1cm electric shock cup, once (1.8KV, 3.4ms) of electric shock under the corresponding program of electroporation, adds rapidly 1ml LB substratum, after mixing, be transferred in 1.5ml centrifuge tube, in 37 DEG C, 150rpm cultivates 1hr, gets appropriate bacterium liquid and coats LB (Amp+) flat board.Be placed in 37 DEG C of incubators, overnight incubation.
Picking mono-clonal access 1mL LB liquid nutrient medium from transforming flat board, 37 DEG C, 220rpm cultivates 6-8hr, carry out bacterium liquid PCR screening, after the positive inoculation filtering out, further extract in a small amount plasmid, and carry out enzyme and cut qualification, enzyme is cut qualification and is sent to company's order-checking after errorless, and the pPink α-TMP-L1-TMP-L2-H-fip-HSA plasmid that checks order correct is clone No. 1.
Embodiment 2: the conversion of recombinant vectors, screening, qualification
The Screening and Identification of expression vector adopts following method conventionally: first by pichia spp recombinant plasmid, pPink α-TMP-L1-TMP-L2-H-fip-HSA, use restriction enzyme linearizing, proceeded in pichia spp by electrotransformation, coated plate is cultivated 3-7 days, chooses 3-8 white clone and is inoculated into (formula reference " PichiaPink in liquid B MGY substratum
tMexpression System "), (formula is with reference to " PichiaPink to change BMMY liquid nutrient medium
tMexpression System ") continue shake-flask culture 3-4 days, obtain a small amount of expression product, now can pass through PCR, SDS-PAGE or Western blot (concrete grammar is shown in embodiment 4) carry out screening and identification to expression product; Then the bacterial strain that contains suitable positive object band filtering out is carried out to fermentation culture conditions optimization, thereby realize the high efficient expression of the Dimerized thrombopoietin simulating peptide of recombinant yeast pichia pastoris TMP diad-human serum albumin fusion proteins.Operation steps can be referring to PichiaPink in detail
tMexpression System (Invitrogen, USA) operational guidance, it is as follows that the electricity of expression vector transforms concrete grammar:
1) a large amount of recombinant plasmid pPink α-TMP-L1-TMP-L2-H-fip-HSA plasmid DNA that build of extracting, then choose restriction enzyme A flII (NEB, USA) plasmid are carried out to linearizing, and purifying is quantitatively rear for subsequent use.
2) prepare yeast competence: by PichiaPink
tMexpression Strains (invitrogen, USA) lines YPD flat board, cultivates 3-5 days for 28 DEG C.After growing clone, picking mono-clonal enters 28 DEG C of 10ml YPD substratum, and 300rpm cultivates 1-2 days, gets 1ml culture access 100miYPD substratum, and the same terms is cultured to OD
600=1.3-1.5.Culture is transferred in 250ml centrifuge tube, and 4 DEG C, the centrifugal 5min of 1500 × g, abandons supernatant, and precipitation is resuspended in 250ml frozen water.Repeat previous step recentrifuge, abandon supernatant, precipitation is resuspended in 50ml frozen water.Repeat the continuation of previous step condition centrifugal, abandon supernatant, precipitation is resuspended in the ice sorbyl alcohol of 10ml1M.Repeat the continuation of previous step condition centrifugal, abandon supernatant, precipitation is resuspended in the ice sorbyl alcohol of 300 μ l1M.The yeast competence preparing is placed on ice, and used the same day.
3) pPink α-TMP-L1-TMP-L2-H-fip-HSA plasmid DNA of getting after 10 μ g linearizings is mixed with the yeast strain competent cell preparing, and is transferred in the electric shock cup of 0.2cm.Put 5min on ice, under program corresponding to electroporation, electric shock once.Add immediately the YPDS of 1ml ice bath, after mixing, put into 28 DEG C of incubators and leave standstill cultivation 6hr.Go appropriate bacteria suspension to coat PAD flat board, cultivate 3-10 days for 28 DEG C.After growing obvious clone, the multiple white clones of picking line respectively PAD flat board again.
White clone after the line of picking secondary is inoculated in 50ml BMGY substratum respectively, and 28 DEG C, 300rpm, cultivates 3 days.
After centrifugal bacterium liquid, abandon supernatant, with the resuspended thalline of 10ml BMMY substratum, 28 DEG C, 300rpm, cultivates 72-96hr.
Sample 500 μ l every 12hr, and add 500 μ l40% methyl alcohol.After sampling, remaining cell continues to cultivate, and sample is in the centrifugal 10min of 1500 × g, and supernatant, precipitation are stored in respectively-80 DEG C, in order to subsequent detection.
When induction finishes, collect all cells, the centrifugal 10min of 1500 × g, supernatant, precipitation are stored in respectively-80 DEG C.
Supernatant, the deposit sample of each time point before above-mentioned all each clones' induction and after induction carried out to reductibility SDS-PAGE electrophoresis detection, only have the supernatant sample after methanol induction to have object band at 75kDa place, and with the increase of abduction delivering time, band object content increases.Select the highest clone of object expression amount to be No. 1 yeast strain with TMP-L1-TMP-L2-H-fip-HSA sequence of acquisition.
Embodiment 3 restructuring have the fermentation of the pichia spp of TMP-L1-TMP-L2-H-fip-HSA sequence
YPD liquid nutrient medium (taking 1L as example): 10g yeast extract, 20g peptone, 20g glucose.
BMGY substratum (taking 1L as example): 10g yeast extract, 20g peptone, the phosphoric acid buffer of the 1MpH6.0 of 100ml, 13.4g is without amino acid yeast basis nitrogenous source substratum, 0.004g vitamin H, 10g100% glycerine.
BMMY substratum (taking 1L as example): 10g yeast extract, 20g peptone, the phosphoric acid buffer of the 1M pH6.0 of 100ml, 13.4g is without amino acid yeast basis nitrogenous source substratum, 0.004g vitamin H, 5ml100% methyl alcohol.
Basic salt culture medium (taking 1L as example) ferments: 40g100% glycerine, 26.7ml85%v/v phosphoric acid, 0.93g calcium sulfate, 18.2g potassium sulfate, 14.9g bitter salt, 4.13g potassium hydroxide.
PTM
1(taking 1L as example): 6.0g Salzburg vitriol, 0.08g sodium iodide, 3.0g magnesium sulfate, 0.2g Sodium orthomolybdate, 0.02g boric acid, 0.5g cobalt chloride, 20g zinc chloride, 65g ferrous sulfate, 0.2g vitamin H, 5ml sulfuric acid.
The present invention also provides the method for utilizing the high efficient expression of described pichia spp PichiaPinkTM Expression Strains and producing the Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins of coding, comprising with the next stage:
A: preparation seed liquor: picking be kept at-20 DEG C No. 1 pichia spp list bacterium colony, be inoculated in 10mLYPD liquid nutrient medium, 30 DEG C of shaking culture are spent the night, transfer in 100mLYPD liquid nutrient medium with 10% inoculum size, 30 DEG C of shaking culture 24 hours, then transfer in 1L BMGY substratum with the inoculum size of 10%v/v, 30 DEG C of shaking culture 16-24 hour, while reaching OD600=5-20 to the yeast amount in fermented liquid, stop fermentation, above-mentioned fermented liquid is as the seed liquor of ferment tank.
B: yeast culture: held 7L basis salt fermention medium in 20L fermentor tank, contained 40g100% glycerine, 26.7ml85%v/v phosphoric acid in the salt fermention medium of every 1L basis, 0.93g calcium sulfate, 18.2g potassium sulfate, 14.9g bitter salt, 4.13g potassium hydroxide, cultivates 20-28 hour.
C: stream adds carbon source: by peristaltic pump flow feeding liquid, the aqueous glycerin solution that feed supplement liquid is 50%v/v, wherein contains 12ml PTM1/L, and stream dosage is 10ml/hr/L, 2-8 hour are cultivated in 30 DEG C of aeration-agitations, reach 180-220g/L to fermented liquid thalline weight in wet base;
D: abduction delivering: pulsed stream adds 100% methyl alcohol in three stages, contains 12ml/L PTM1 in described methyl alcohol, ensure that the dissolved oxygen amount in fermented liquid is greater than 20%, 26-28 DEG C of aeration-agitation cultivation 60-96 hour all the time:
First stage: 2ml/hr/L stream of pulses adds 100% methyl alcohol 8 hours, in described methyl alcohol, contain 12ml PTM1/L, this stage adds and adjusts fermented liquid pH at 5.90-6.00 with the citric acid stream of 0.5M.
Subordinate phase: 4ml/hr/L stream of pulses adds 100% methyl alcohol 4 hours, contains 12ml PTM1/L in described methyl alcohol, this stage adds and adjusts fermented liquid pH at 5.90-6.00 with 50%v/v ammoniacal liquor stream.
Phase III: 8ml/hr/L stream of pulses adds 100% methyl alcohol 48-84 hour to fermentation ends, contains 12ml PTM1/L in described methyl alcohol, this stage adds and adjusts fermented liquid pH at 5.90-6.00 with 50%v/v ammoniacal liquor stream.
Control group is same adopts No. 1 Pichi strain to ferment, and has adopted general fermentation strategies in " Pichia Fermentation Process Guidelines " to carry out the abduction delivering of target protein:
A: preparation seed liquor: picking be kept at-20 DEG C No. 1 pichia spp list bacterium colony, be inoculated in 10mL YPD liquid nutrient medium, 30 DEG C of shaking culture are spent the night, transfer in 100mLYPD liquid nutrient medium with the inoculum size of 10%v/v, 30 DEG C of shaking culture 24 hours, transfer in 1L BMGY substratum with the inoculum size of 10%v/v again, 30 DEG C of shaking culture 16-24 hour, while reaching OD600=5-20 to the yeast amount in fermented liquid, stop fermentation, above-mentioned fermented liquid is as the seed liquor of ferment tank.
B: yeast culture: held 7L basis salt fermention medium in 20L fermentor tank, contained 40g100% glycerine, 26.7ml85%v/v phosphoric acid in the salt fermention medium of every 1L basis, 0.93g calcium sulfate, 18.2g potassium sulfate, 14.9g bitter salt, 4.13g potassium hydroxide, cultivates 20-28 hour.
C: stream adds carbon source: by peristaltic pump flow feeding liquid, the aqueous glycerin solution that feed supplement liquid is 50%v/v, wherein contains 12ml PTM1/L, and stream dosage is 10ml/hr/L, 2-8 hour are cultivated in 30 DEG C of aeration-agitations, reach 180-220g/L to fermented liquid thalline weight in wet base;
D: abduction delivering: pulsed stream adds 100% methyl alcohol in three stages, contains 12ml/L PTM1 in described methyl alcohol, ensure that the dissolved oxygen amount in fermented liquid is greater than 20%, 26-28 DEG C of aeration-agitation cultivation 60-96 hour all the time:
First stage: 2ml/hr/L stream of pulses adds 100% methyl alcohol 4 hours, in described methyl alcohol, contain 12ml PTM1/L.
Subordinate phase: 4ml/hr/L stream of pulses adds 100% methyl alcohol 4 hours, contains 12ml PTM1/L in described methyl alcohol.
Phase III: 8ml/hr/L stream of pulses adds 100% methyl alcohol 52-88 hour to fermentation ends, contains 12ml PTM1/L in described methyl alcohol.
Above-mentioned stream adds the stage and all uses 50%v/v ammoniacal liquor stream to add the pH value of adjusting fermented liquid at 5.90-6.00.
As shown in Figure 2 and Figure 3, in experimental group, No. 1 pichia spp biomass raises fermentation results gradually; And in control group, the biomass of No. 1 pichia spp raise gradually in the glycerine increasing bacterium stage, and reduce gradually at methanol induction expression phase.
Embodiment 4Western blotting testing goal albumen
Get the object product (control sample, laboratory sample) after purifying to be detected, carry out Westernblotting detection.
1) conventional SDS-PAGE method, concrete steps are omitted.
2) transferring film: prepare 4 filter paper, a nitrocellulose filter according to the size of the target protein region blob of viscose of albumen Maker instruction.Electricity consumption turns immersion bubble filter paper, film and electricity and turns the sponge in folder.After electrophoresis finishes, cut the blob of viscose that contains target protein, clip electricity turn folder according to the order of sponge-two filter paper-nitrocellulose filter-blob of viscose-two filter paper-sponge, film is in an anodal side.In electricity turn trough, place ice block cooling, 300mA, 1 hour.
3) sealing: film is taken out from electric turn trough, and TTBS rinsing 30min, is immersed in confining liquid (1%w/v casein) and slowly sways 2 hours
4) in conjunction with primary antibodie: film is immersed in the sealing plastic film that primary antibodie (Anti-HSA/human, 1: 1000, the raw work in Shanghai) is housed, and under room temperature, jog is hatched 1 hour or 4 DEG C of hold over night
5) washing: primary antibodie is hatched after end, with TTBS rinsing three times, each 5-10min.
6) anti-in conjunction with two: film is immersed in the sealing plastic film that two anti-(horseradish enzyme labelling goat anti-mouse IgG, 1: 1000, biotech firms of Zhong Shan Golden Bridge) are housed, and under room temperature, jog is hatched 1 hour
7) washing: two anti-hatching after end, use TTBS rinsing film three times, each 5-10min.
8) DAB colour developing.Operate according to DAB staining kit (Beijing Bo Aosen Bioisystech Co., Ltd) step.
As shown in Figure 4, Figure 5, Fig. 4 shows and in experimental group tunning, detected object product (TMP-L1-TMP-L2-H-fip-HSA) detected result, and Fig. 5 shows and in control group tunning, do not detect object product.Fermentation results and object product detected result all show, in the time adopting fermentation strategies of the present invention to carry out the fermentation of No. 1 Pichi strain, and the growth that bacterial strain can fast and stable in high methanol environment, thalline weight in wet base obviously raises, and gives expression to target protein.And in the time adopting the fermentation strategies of classical invitrogen to ferment, No. 1 Pichi strain cannot be grown in the methyl alcohol environment of high density, thalline weight in wet base reduces gradually, occurs the situation of mortality, and the target protein that is beyond expression.
Claims (6)
1. the DNA sequence dna of the Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins of coding, is characterized in that, its nucleotide sequence is as shown in SeqID No.1.
2. Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins, is characterized in that, its aminoacid sequence is as shown in SeqIDNo.2.
3. the DNA sequence dna of the Dimerized thrombopoietin simulating peptide of coding according to claim 1 TMP diad-human serum albumin fusion proteins, it is characterized in that this sequence be reconstituted in pPink α-HC or other can abduction delivering and secrete in the plasmid vector of recombinant protein outside born of the same parents after, this recombinant plasmid vector is integrated in yeast host cell karyomit(e).
4. encode the according to claim 3 DNA sequence dna of Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins, is characterized in that this yeast host cell is Pichi strain PichiaPink
tMexpression Strains or other can expression alien gene yeast strain.
5. a pichia spp PichiaPink
tMthe high efficient expression of Expression Strains and the method for producing the Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins of coding, comprise that glycerine increases bacterium stage and methanol induction expression phase, it is characterized in that: at the initial stage of methanol induction expression phase, add and adjust fermented liquid pH at 5.90-6.00 with the citric acid stream of 0.5M.
6. a kind of pichia spp PichiaPink according to claim 5
tMthe high efficient expression of Expression Strains and the method for producing the Dimerized thrombopoietin simulating peptide TMP diad-human serum albumin fusion proteins of coding, it is characterized in that: at the initial stage of methanol induction expression phase, the flow acceleration of 100% methyl alcohol is 2ml/hr/L, it is 8 hours that stream adds the time, contains 12ml PTM1/L in methyl alcohol.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310084035.3A CN104046642B (en) | 2013-03-15 | 2013-03-15 | The method of the Dimerized fusion protein of fermenting and producing |
| PCT/CN2014/077618 WO2014187269A1 (en) | 2013-03-15 | 2014-05-15 | Method for producing dimerized fusion protein by fermentation |
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| CN109748969A (en) * | 2017-11-08 | 2019-05-14 | 兰州大学 | A kind of Dimerized fusion protein and its preparation method and application |
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| CN106279398A (en) * | 2015-06-26 | 2017-01-04 | 天津药物研究院有限公司 | A kind of Erythropoietin mimetic peptide and its preparation method and application |
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| CN109748969A (en) * | 2017-11-08 | 2019-05-14 | 兰州大学 | A kind of Dimerized fusion protein and its preparation method and application |
| CN109748969B (en) * | 2017-11-08 | 2023-08-08 | 兰州大学 | Dimerization fusion protein and preparation method and application thereof |
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| CN104046642B (en) | 2018-07-06 |
| WO2014187269A1 (en) | 2014-11-27 |
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