CN103525712B - Yeast transformant of secreting, expressing foreign protein and preparation method thereof - Google Patents
Yeast transformant of secreting, expressing foreign protein and preparation method thereof Download PDFInfo
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Abstract
The invention discloses yeast transformant of a kind of secreting, expressing foreign protein and preparation method thereof, described method steps is: forward primer 5 ' is held and introduced XhoI, 20 seed amino acid codons are introduced in corresponding Kex2 identified region P1 ' site respectively, and reverse primer 5 ' is held and introduced NotI, obtains 20 pairs of primers; With target gene CDS sequence cDNA or DNA for template amplification, TA clones to obtain carrier group P1 '-CDS-pMD20-T; Build yeast carrier for expression of eukaryon group P1 '-CDS-pPICZ α A; Conversion carrier group is to yeast, and screen positive transformant, and receive YPD flat board, obtain the conversion subgroup methanol induction of identical copies number, analyzing proteins output, obtains the transformant that expression level is the highest.Preparation method of the present invention is preferred with the activity improving Kex2 proteolytic enzyme identification substrate by the P1 ' amino-acid residue to Kex2, thus improves the output of yeast secreted expression recombinant protein.
Description
Technical field
The present invention relates to application recombinant DNA technology producer gene engineered protein drug technique, specifically, particularly a kind ofly obtain yeast transformant of foreign protein high-level secretory expression and preparation method thereof by construction of recombinant vector and yeast transformant screening.
Background technology
KEX2 is the member of huge precursor protein saccharase family, and this protein family is relevant to bacteria B bacillin.The homologous protein of Kex2 on pathogenicity bo yeast Candidaalbicans is a virulence factor.The eucaryon homologous protein of this albumen comprises furin, PC1, PC2 and other precursor protein saccharases for the treatment of such as precursor Regular Insulin, precursor hyperglycemic-glycogenolytic factor and precursor neuropeptide etc. secreting hormone.The disappearance of these genes is considered to such as the major disease such as diabetes and cancer is in close relations.
What KEX2 genes encoding one participated in the processing of intracellular protein precursor depends on Ca
2+neutral serine.Kex2 albumen is film binding albumen, and it is positioned endoplasmic reticulum the earliest and finally plays function at trans Golgi network, in trans Golgi vesicles and the cocycle of the endosomal membrane in late period bubble.
Kex2 is expressed out with the precursor forms of a non-activity at first, and this precursor protein contains multiple structural domain, has some finally cutly can fall formed ripe albumen.The structural domain of Kex2 proenzyme comprises: a N-terminal endoplasmic reticulum signal peptide, then a front body structure territory below, a catalyst structure domain, a P-structure territory, Unknown Function is still guarded and is necessary for catalytic activity in the body of this albumen in the precursor protein saccharase family belonging to this albumen, the serine/threonine rich region of a high-glycosylation, a single pass transmembrane structural domain and an afterbody going deep into endochylema containing trans Golgi network signal for locating.In the activation process of Kex2, the N-terminal of this albumen can experience repeatedly posttranslational modification step.First, signal peptide is just cut in endoplasmic reticulum while translation; Then remove by the excision effect of self mediation intramolecular the front body structure territory suppressing son as molecular chaperones and self, this enzyme molecule has just been activated; Also have afterwards and further in golgi body, carry out N-terminal modification by the two peptide aminopeptidase of Ste13, finally generate ripe Kex2.
Kex2 has strict optionally protein incision enzyme as one to substrate sequence, and it is more clearly that its substrate selective has been understood.Here the method for single amino acids residue on a kind of name substrate of being accepted extensively by scientific circles and relevant proteolytic enzyme is first introduced: from digested disconnected peptide bond, N-terminal number toward substrate is gone over, and the amino acid of substrate is denoted as P1 successively, P2, P3, P4 ..., to bind with them and the amino acid of interactional enzyme molecule is denoted as S1 successively, S2, S3, S4, In like manner, the C-terminal number toward substrate is gone over, and the amino acid of substrate is denoted as P1 ' successively, P2 ', P3 ', P4 ' ..., to bind with them and the amino acid of interactional enzyme molecule is denoted as S1 ', S2 ', S3 successively ', S4 ' ...
Though abroad have been reported, good Kex2 activity can bring the secreting, expressing amount of the natural pheromone of higher yeast, and our scheme is on the condition of the optimum level obtained in this report, the optimization carried out further.At present, for Kex2, generally acknowledged substrate specificity is: P1 is strictly special can only identify Arg; P2 identifiable design alkaline amino acid residue, as Lys, Arg and ornithine; P4 identifiable design aliphatics or alkaline amino acid residue; For the other side of cleaved peptide bond, it is generally acknowledged there is no special selectivity, just compare the larger side chain of exclusion volume in P1 ' site; So, do not report the optionally special shape of the P1 ' residue of Kex2, more do not report that this can remarkably influenced yeast secreted expression level, and and then the level of the yeast secreted expression improving recombinant protein greatly can be utilized.
Summary of the invention
Based on this, the invention provides yeast transformant of a kind of high-level secretory expression foreign protein and preparation method thereof, the method, by optimizing yeast Kex2 protease activity and promoting its expression amount, obtains the yeast transformant of the high-level secretory expression of the foreign gene of restructuring.
The concrete technical scheme solved the problems of the technologies described above is as follows:
Obtain a method for the Yeast transformant of foreign protein high-level secretory expression, comprise the following steps:
(1) primer of a series of cloned foreign gene CDS of design and synthesis: the position corresponding to P1 ' at forward primer, i.e. first amino acid in Kex2 shearing site C-terminal direction, introduce the codon of 20 kinds of natural amino acids respectively, forward primer 5 ' holds design XhoI site, region between XhoI site and P1 ' site is filled by the corresponding sequence (AAAAGA) of original yeast secreted expression carrier, P1 ' sites downstream to primer 3 ' end is that PCR reacts extension increasing sequence complementary region, should merge under same expression cassette to the CDS be amplified from XhoI site, 3 ' region and the foreign gene 3 ' termini-complementary be amplified of reverse primer, 5 ' end introduces NotI site for inserting yeast secreted expression carrier, obtain 20 to serial primer,
(2) TA clones interested certain foreign gene CDS: utilize the serial primer described in step (1), the PCR reaction of each forward primer, all to arrange in pairs or groups same reverse primer, with the cDNA containing the complete CDS sequence of target gene or other DNA moleculars for template, amplify complete gene fragment, reclaim PCR primer TA and connect into TA cloning vector pMD20-T, obtain Series P 1 ' and change carrier group P1 '-CDS-pMD20-T, identical with design through sequence verification all carriers P1 ' site amino acids codon, and foreign gene is also correct expressible nucleotide sequence,
(3) sub clone construction yeast carrier for expression of eukaryon: the Series P 1 ' described in step (2) is changed carrier group P1 '-CDS-pMD20-T and yeast secreted expression carrier pPICZ α A, double digested through XhoI and NotI, reclaim Insert Fragment and carrier segments respectively, connect reagent with SolutionI and connect above-mentioned recovery fragment, obtain Series P 1 ' change recombinant vectors group the P1 '-CDS-pPICZ α A that pPICZ α A recombinates;
(4) recombination yeast secretion expression carrier is transformed in yeast host: by recombinant vectors group the P1 '-CDS-pPICZ α A of step (3) gained after the linearizing of SacI single endonuclease digestion, reclaim digestion products, proceed in the X-33 Host Strains of Pichia pastoris through lithium chloride conversion method, positive transformant is obtained, with PCR reaction checking positive transformant through zeocin90-110 μ g/ml screening;
(5) yeast transformant copy number is determined: be transferred to by the positive yeast transformant of step (4) gained on YPD flat board that the concentration level containing zeocin increases progressively, for determining the exogenous origin gene integrator copy number of transformant, allly can not be considered as that there is identical exogenous origin gene integrator copy number at the yeast transformant of next greater concn horizontal growth at the YPD grow on plates of same zeocin concentration level, each recombinant vectors yeast transformant determines each 5-6 of yeast clone with anti-identical zeocin concentration level, specific P1 ' the amino acid whose recombinant vectors transformant that screening secreting, expressing amount is the highest must change at each P1 ' that copy number is identical to compare between serial carrier transformant P1 '-CDS reaches a conclusion,
(6) liquid culture of yeast transformant and methanol induction are expressed: by step (5) gained have the P1 ' of identical exogenous origin gene integrator copy number to change to transform subgroup P1 '-CDS and the contrast of unloaded transformant is seeded to BMGY liquid nutrient medium, be cultured to OD in 25-30 DEG C of shaking table
600for 1.8-2.2, collect yeast cell, and resuspended in BMMY liquid nutrient medium, be diluted to OD
600value is 0.8-9-1.1, and continues to cultivate at 25-30 DEG C of shaking table to induce external source recombination secreting, expressing, and centrifugal yeast culture collects supernatant;
(7) the highest recombinant vectors transformant of expression level is screened: by the output of foreign recombinant proteins in analytical procedure (6) gained supernatant, obtain the amino acid whose recombinant vectors transformant of the highest specific P1 ' of output.
In certain embodiments, described in step (1), the forward primer of the codon of 20 kinds of natural amino acids is:
SEQIDNO.1：
Glu(E):5’-CTCGAGAAAAGAGAA-CDS-3’；
SEQIDNO.2：
Ala(A):5’-CTCGAGAAAAGAGCT-CDS-3’；
SEQIDNO.3：
Arg(R):5’-CTCGAGAAAAGAAGA-CDS-3’；
SEQIDNO.4：
Asn(N):5’-CTCGAGAAAAGAAAC-CDS-3’；
SEQIDNO.5：
Asp(D):5’-CTCGAGAAAAGAGAT-CDS-3’；
SEQIDNO.6：
Cys(C):5’-CTCGAGAAAAGATGT-CDS-3’；
SEQIDNO.7：
Gln(Q):5’-CTCGAGAAAAGACAA-CDS-3’；
SEQIDNO.8：
Gly(G):5’-CTCGAGAAAAGAGGT-CDS-3’；
SEQIDNO.9：
His(H):5’-CTCGAGAAAAGACAT-CDS-3’；
SEQIDNO.10：
Ile(I):5’-CTCGAGAAAAGAATT-CDS-3’；
SEQIDNO.11：
Leu(L):5’-CTCGAGAAAAGACTT-CDS-3’；
SEQIDNO.12：
Lys(K):5’-CTCGAGAAAAGAAAA-CDS-3’；
SEQIDNO.13：
Met(M):5’-CTCGAGAAAAGAATG-CDS-3’；
SEQIDNO.14：
Phe(F):5’-CTCGAGAAAAGATTT-CDS-3’；
SEQIDNO.15：
Pro(P):5’-CTCGAGAAAAGACCA-CDS-3’；
SEQIDNO.16：
Ser(S):5’-CTCGAGAAAAGATCT-CDS-3’；
SEQIDNO.17：
Thr(T):5’-CTCGAGAAAAGAACT-CDS-3’；
SEQIDNO.18：
Trp(W):5’-CTCGAGAAAAGATGG-CDS-3’；
SEQIDNO.19：
Tyr(Y):5’-CTCGAGAAAAGATAT-CDS-3’；
SEQIDNO.20：
Val(V):5’-CTCGAGAAAAGAGTT-CDS-3’。
Wherein in some embodiments, the concentration of zeocin described in step (4) is 100 μ g/ml.
Wherein in some embodiments, the concentration of the zeocin of YPD flat board described in step (5) is followed successively by 200 μ g/ml, 500 μ g/ml and 1000 μ g/ml.
According to above-mentioned preparation method and obtainable yeast transformant.
Yeast transformant of a kind of high-level secretory expression foreign protein of the present invention and preparation method thereof has the following advantages and beneficial effect:
(1) preparation method of the present invention, change the mode of traditional screening yeast recombination high level expression transformant, explore and utilize Kex2 proteolytic enzyme to show different P1 ' optionally feature for different substrate, by building a series of carrier and yeast transformant screens the suitableeest P1 ' amino-acid residue of Kex2 for a certain specific recombination, thus obtain the method for optimum recombinant protein secreting, expressing amount.
(2) preparation method of the present invention is particularly useful for some foreign gene Direct Cloning situation that secreting, expressing recombinant protein amount is lower on original yeast secretion type carrier.
(3) preparation method of the present invention is based on through commercialization optimization, can the yeast secreted expression system of a lot of external source recombination of efficient secretory expression, the further optimization that the basis of this system is done, jointly can use with original raising yeast foreign gene secreting, expressing means, original expression level improves output further.
Accompanying drawing explanation
Fig. 1 is that embodiment 1-6 yeast carrier for expression of eukaryon Series P 1 ' change carrier group P1 '-Venus/Luciferase/KITligand/FGF16/FGF20/Irisin-pPICZ α A builds schematic diagram;
Fig. 2 is that the Series P 1 ' of concentration 200 μ g/mlzeocin resistance level changes yeast transformant P1 '-Venus abduction delivering day part fluorescent signal OD
600value;
Fig. 3 is that the Series P 1 ' of concentration 500 μ g/mlzeocin resistance level changes yeast transformant P1 '-Venus abduction delivering day part fluorescent signal OD
600value;
Fig. 4 is that the Series P 1 ' of concentration 200 μ g/mlzeocin resistance level changes yeast transformant P1 '-Luciferase abduction delivering day part chemiluminescence signal OD
600value;
Fig. 5 is that the Series P 1 ' of anti-500 μ g/mlzeocin resistance levels changes yeast transformant P1 '-Luciferase abduction delivering day part chemiluminescence signal OD
600value.
Embodiment
The Yeast transformant preparation method of a kind of high-level secretory expression foreign protein of the present invention, be by optimize business-like, can the yeast secreted expression system of a lot of external source recombination of efficient secretory expression, P1 ' the amino-acid residue of preferred Kex2 to improve Kex2 protein incision enzyme identification substrate active, thus improves heterologous protein secretion output further.
CDS of the present invention refers to protein coding region.
Below with reference to specific embodiment, the present invention will be further described.
Pichia pastoris X-33 bacterial strain selected by the present invention, expression vector pPICZ α A equal purchased from American Invitrogen company.
Used medium formula is as follows:
1) BMGY substratum (yeast growth medium):
Dissolve 10g yeast extract completely, 20g peptone, constant volume is to 700mL.121 DEG C of steam autoclaving 15-20min, cool to room temperature, adds 100mL1M potassium phosphate solution, 100mLYNB, 2mL500*B, 100mL10*GY;
2) BMMY substratum (yeast inducing culture):
Dissolve 10g yeast extract completely, 20g peptone, constant volume is to 700mL.121 DEG C of steam autoclaving 15-20min, cool to room temperature, adds 100mL1M potassium phosphate solution, 100mLYNB, 2mL500*B, 100mL10*M;
3) YPD substratum
Dissolve 10g yeast extract completely, 20g peptone, 10g glucose, constant volume to 1000mL, 121 DEG C of steam autoclaving 15-20min;
Above-mentioned YPD substratum, if desired uses solid medium, then must add 15g agar in 1000mL liquid nutrient medium.
Yeast transformant of embodiment 1 one kinds of high-level secretory expression Venus albumen and preparation method thereof
The preparation method of the Yeast transformant of secreting, expressing foreign protein, comprises the following steps:
(1) primer of a series of clone Venus gene is designed:
Forward primer introduces XhoI site at 5 ' end, the codon of 20 kinds of natural amino acids is introduced respectively in the P1 ' site corresponding to Kex2 proteolytic enzyme identified region, region between XhoI site with P1 ' site is filled by the corresponding sequence (AAAAGA) of original yeast secreted expression carrier, P1 ' sites downstream to primer 3 ' end is that PCR reacts extension increasing sequence complementary region, should merge under same expression cassette to the Venus gene be amplified from XhoI site, 3 ' region and the foreign gene 3 ' termini-complementary be amplified of reverse primer, 5 ' end of reverse primer introduces NotI site for inserting yeast secreted expression carrier, obtain 20 to serial primer,
Described 20 forward primers comprising the codon of 20 kinds of natural amino acids are respectively:
SEQIDNO.21：
Glu(E):5’-CTCGAGAAAAGAGAA
ATGGTGAGCAAGGGCGAGGAGCTG-3’；
(introduce XhoI site, underscore part is that VenusCDS5 ' holds complementary region)
SEQIDNO.22：
Ala(A):5’-CTCGAGAAAAGAGCTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.23：
Arg(R):5’-CTCGAGAAAAGAAGAATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.24：
Asn(N):5’-CTCGAGAAAAGAAACATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.25：
Asp(D):5’CTCGAGAAAAGAGATATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.26：
Cys(C):5’-CTCGAGAAAAGATGTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.27：
Gln(Q):5’-CTCGAGAAAAGACAAATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.28：
Gly(G):5’-CTCGAGAAAAGAGGTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.29：
His(H):5’-CTCGAGAAAAGACATATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.30：
Ile(I):5’-CTCGAGAAAAGAATTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.31：
Leu(L):5’-CTCGAGAAAAGACTTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.32：
Lys(K):5’-CTCGAGAAAAGAAAAATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.33：
Met(M):5’-CTCGAGAAAAGAATGATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.34：
Phe(F):5’-CTCGAGAAAAGATTTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.35：
Pro(P):5’-CTCGAGAAAAGACCAATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.36：
Ser(S):5’-CTCGAGAAAAGATCTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.37：
Thr(T):5’-CTCGAGAAAAGAACTATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.38：
Trp
(W):5’-CTCGAGAAAAGATGGATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.39：
Tyr
(Y):5’-CTCGAGAAAAGATATATGGTGAGCAAGGGCGAGGAGCTG-3’；
SEQIDNO.40：
Val
(V):5’-CTCGAGAAAAGAGTTATGGTGAGCAAGGGCGAGGAGCTG-3’；
The unique reverse primer matched with above-mentioned 20 forward primers is:
SEQIDNO.41:5 '-GCGGCCGC
cTTGTACAGCTCGTCCATGCCGAG-3 ' (introduce NotI site, underscore part is that VenusCDS3 ' holds complementary region)
(2) TA clones interested Venus gene:
Utilize the interested target protein gene of the serial primer amplification of 20 couple described in step (1).
Wherein, PCR condition is 95 DEG C of denaturations, 5min, a thermal cycling; 95 DEG C of thermally denature 30s, 55 DEG C of annealing 30s, 72 DEG C of extension times determine depending on target dna sequence length, average 1kb/min, 30 thermal cyclings; 72 DEG C of renaturation 10min.The amplified fragments obtained is connected to pMD20-T carrier (purchased from Dalian TaKaRa company), recombinant vectors is through order-checking, checking P1 ' amino acid codes and target gene sequence correctly complete, obtain Series P 1 ' and change carrier group P1 '-Venus-pMD20-T.
(3) sub clone construction yeast carrier for expression of eukaryon group P1 '-Venus-pPICZ α A:
1. use the double digested Series P 1 ' of XhoI and NotI to change carrier group P1 '-Venus-pMD20-T, reclaim object fragment the P1 '-CDS inserted, reaction system following (restriction endonuclease used and damping fluid are all purchased from Dalian TaKaRa company):
2. use XhoI and NotI double digested pPICZ α A, reclaim carrier segments, reaction system following (restriction endonuclease used and damping fluid are all purchased from Dalian TaKaRa company):
3. by 1. and 2. walking gained object fragment and carrier segments, DNA gel is regained test kit and is reclaimed, and this test kit is purchased from Dalian TaKaRa company, and concrete operations are undertaken by test kit specification sheets.
4. reclaim the object fragment obtained to be connected reagent (purchased from Dalian TaKaRa company) with carrier with SolutionI and to carry out ligation, goal gene is accurately inserted in the excretion vector reading frame containing secretion signal α-factor, and reaction system is as follows:
Plasmid pPICZ α A carrier segments 1 μ L
Object fragment 4 μ L
SolutionI connects reagent 5 μ L
The serial P1 ' that must recombinate changes yeast carrier for expression of eukaryon group P1 '-Venus-pPICZ α A, builds schematic diagram and sees Fig. 1.
(4) recombination yeast secretion expression carrier is transformed in yeast host:
By LiCl yeast conversion legal system for competent yeast, 5-10 μ g volume after the Series P 1 ' of checking changes the linearizing of yeast carrier for expression of eukaryon group P1 '-Venus-pPICZ α A SacI single endonuclease digestion is adjusted to 50 μ L, experimental procedure according to LiCl yeast conversion method adds competent yeast cells and other reagent, is transformed into by serial recombinant vectors (LiCl yeast conversion method concrete steps are see the EasySelectTMPichiaExpressionKit specification sheets of Invitrogen company) in host's Pichia yeast X-33; Obtaining positive transformant with carrying out screening containing the antibiotic YPD flat board of 100 μ g/mLzeocin after transforming, with PCR, transformant being verified.
(5) yeast transformant copy number is determined:
Positive yeast transformant is transferred to containing 200, 500 and 1000 μ g/mlzeocin flat boards are determined the exogenous origin gene integrator copy number of transformant, allly can not be considered as that there is identical exogenous origin gene integrator copy number at the yeast transformant of next greater concn horizontal growth at the YPD grow on plates of same zeocin concentration level, each recombinant vectors yeast transformant determines each 5-6 of yeast clone with identical zeocin concentration level, specific P1 ' the amino acid whose recombinant vectors transformant that screening secreting, expressing amount is the highest must change at each P1 ' that copy number is identical to compare between serial carrier transformant reaches a conclusion.
(6) liquid culture of yeast transformant and methanol induction are expressed:
P1 ' the change determining step (5) to have identical exogenous origin gene integrator copy number transforms subgroup P1 '-Venus and the contrast of unloaded transformant is seeded to BMGY liquid nutrient medium, and 25-30 DEG C of shaking table is cultured to OD
600measured value about 2, collects yeast cell and (is diluted to OD so that BMMY liquid nutrient medium is resuspended
600value is about 1) and continue to cultivate at 25-30 DEG C of shaking table to induce external source recombination secreting, expressing, detailed methanol induction Pichiapastoris foreign gene secreting, expressing operation steps is see the EasySelectTMPichiaExpressionKit specification sheets of Invitrogen company.From start with BMMY induction (0h), receive a sample every 24h, coinduction 120h, the yeast culture matter sample collected by centrifugation supernatant of results.
(7) the high recombinant vectors transformant of expression level is screened
Detect the fluorescent signal that Venus genetic expression produces, to determine the secreting, expressing level of Venus gene.
Detection method: 80 μ L yeast culture supernatants are added in each hole of the real end 96 orifice plate (purchased from American PerkinElmer company) of CulturPlateTM-96 black, carry out fluorescent signal detection under the excitation wavelength of 515nm and the determined wavelength condition of 528nm.By comparing, filter out the amino acid whose recombinant vectors transformant of the highest specific P1 ' of output, result is see Fig. 3-Fig. 4.
In addition to the above methods, also can with ELISA or Westernblotting direct-detection target protein expression amount.
From Fig. 2 and Fig. 3, different P1 ' amino-acid residues causes the secreting, expressing level being integrated into the restructuring foreign gene Venus of Yeast genome with secretion expression carrier pPICZ α A to produce sizable difference; And the difference of copy number, significantly can not change the difference (high is still high, and low is still low) of the relative expression levels that each P1 ' amino-acid residue causes, each transformed yeast strain secreting, expressing level only can be made to be similar to and change on year-on-year basis.For Venus, Serine (Ser) can show the highest secreting, expressing level when P1 ' site, is equivalent to about 13 times of the tyrosine (Tyr) of minimum level.Just can choose the highest Ser-Venus bacterial strain of secreting, expressing amount accordingly, in order to express this foreign protein on a large scale.
The protein-active that the activity of the Venus albumen obtained by the present embodiment is obtained with ordinary method is substantially identical.
Yeast transformant of embodiment 2 one kinds of high-level secretory expression luciferase albumen and preparation method thereof
The preparation method of the Yeast transformant of secreting, expressing foreign protein, comprises the following steps:
(1) primer of a series of clone luciferase gene is designed:
Forward primer introduces XhoI site at 5 ' end, the codon of 20 kinds of natural amino acids is introduced respectively in the P1 ' site corresponding to Kex2 proteolytic enzyme identified region, region between XhoI site with P1 ' site is filled by the corresponding sequence (AAAAGA) of original yeast secreted expression carrier, P1 ' sites downstream to primer 3 ' end is that PCR reacts extension increasing sequence complementary region, should merge under same expression cassette to the luciferase gene be amplified from XhoI site, 3 ' region and the foreign gene 3 ' termini-complementary be amplified of reverse primer, 5 ' end of reverse primer introduces NotI site for inserting yeast secreted expression carrier, obtain 20 to serial primer,
Described 20 forward primers comprising the codon of 20 kinds of natural amino acids are respectively:
SEQIDNO.42：
Glu
(E): 5 '-CTCGAGAAAAGAGAA
aTGGAAGACGCCAAAAACATAAAG-3 ' (introduce XhoI site, underscore part is that LuciferaseCDS5 ' holds complementary region);
SEQIDNO.43：
Ala(A):5’-CTCGAGAAAAGAGCTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.44：
Arg(R):5’-CTCGAGAAAAGAAGAATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.45：
Asn(N):5’-CTCGAGAAAAGAAACATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.46：
Asp(D):5’-CTCGAGAAAAGAGATATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.47：
Cys(C):5’-CTCGAGAAAAGATGTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.48：
Gln(Q):5’-CTCGAGAAAAGACAAATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.49：
Gly(G):5’-CTCGAGAAAAGAGGTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.50：
His(H):5’-CTCGAGAAAAGACATATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.51：
Ile(I):5’-CTCGAGAAAAGAATTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.52：
Leu(L):5’-CTCGAGAAAAGACTTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.53：
Lys(K):5’-CTCGAGAAAAGAAAAATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.54：
Met(M):5’-CTCGAGAAAAGAATGATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.55：
Phe(F):5’-CTCGAGAAAAGATTTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.56：
Pro(P):5’-CTCGAGAAAAGACCAATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.57：
Ser(S):5’-CTCGAGAAAAGATCTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.58：
Thr(T):5’-CTCGAGAAAAGAACTATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.59：
Trp(W):5’-CTCGAGAAAAGATGGATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.60：
Tyr(Y):5’-CTCGAGAAAAGATATATGGAAGACGCCAAAAACATAAAG-3’；
SEQIDNO.61：
Val(V):5’-CTCGAGAAAAGAGTTATGGAAGACGCCAAAAACATAAAG-3’。
The described unique reverse primer matched with above-mentioned 20 forward primers is:
SEQIDNO.62:5 '-GCGGCCGCCACGGCGATCTTTCCGCCCTTC-3 ' (introduce NotI site, underscore part is that LuciferaseCDS3 ' holds complementary region);
(2) TA clones interested Luciferase gene:
Utilize the interested target protein gene of 20 serial primer amplifications described in step (1).
Wherein, PCR condition is 95 DEG C of denaturations, 5min, a thermal cycling; 95 DEG C of thermally denature 30s, 55 DEG C of annealing 30s, 72 DEG C of extension times determine depending on target dna sequence length, average 1kb/min, 30 thermal cyclings; 72 DEG C of renaturation 10min.The amplified fragments obtained is connected to pMD20-T carrier (purchased from Dalian TaKaRa company), recombinant vectors is through order-checking, checking P1 ' amino acid codes and target gene sequence correctly complete, obtain Series P 1 ' and change carrier group P1 '-luciferase-pMD20-T.
(3) sub clone construction yeast carrier for expression of eukaryon group P1 '-luciferase-pPICZ α A:
1. use the double digested Series P 1 ' of XhoI and NotI to change carrier group P1 '-luciferase-pMD20-T, reclaim object fragment the P1 '-CDS inserted, reaction system following (restriction endonuclease used and damping fluid are all purchased from Dalian TaKaRa company):
2. use XhoI and NotI double digested pPICZ α A, reclaim carrier segments, reaction system following (restriction endonuclease used and damping fluid are all purchased from Dalian TaKaRa company):
3. by 1. and 2. walking gained object fragment and carrier segments, DNA gel is regained test kit and is reclaimed, and this test kit is purchased from Dalian TaKaRa company, and concrete operations are undertaken by test kit specification sheets.
4. reclaim the object fragment obtained to be connected reagent (purchased from Dalian TaKaRa company) with carrier with SolutionI and to carry out ligation, goal gene is accurately inserted in the excretion vector reading frame containing secretion signal α-factor, and reaction system is as follows:
Plasmid pPICZ α A carrier segments 1 μ L
Object fragment 4 μ L
SolutionI connects reagent 5 μ L
The serial P1 ' that must recombinate changes yeast carrier for expression of eukaryon group P1 '-luciferase-pPICZ α A, builds schematic diagram and sees Fig. 1.
(4) recombination yeast secretion expression carrier is transformed in yeast host:
By LiCl yeast conversion legal system for competent yeast, 5-10 μ g volume after the Series P 1 ' of checking changes the linearizing of yeast carrier for expression of eukaryon group P1 '-luciferase-pPICZ α A SacI single endonuclease digestion is adjusted to 50 μ L, experimental procedure according to LiCl yeast conversion method adds competent yeast cells and other reagent, is transformed into by serial recombinant vectors (LiCl yeast conversion method concrete steps are see the EasySelectTMPichiaExpressionKit specification sheets of Invitrogen company) in host's Pichia yeast X-33; Obtaining positive transformant with carrying out screening containing the antibiotic YPD flat board of 100 μ g/mLzeocin after transforming, with PCR, transformant being verified.
(5) yeast transformant copy number is determined:
Positive yeast transformant is transferred to containing 200, 500 and 1000 μ g/mlzeocin flat boards are determined the exogenous origin gene integrator copy number of transformant, allly can not be considered as that there is identical exogenous origin gene integrator copy number at the yeast transformant of next greater concn horizontal growth at the YPD grow on plates of same zeocin concentration level, each recombinant vectors yeast transformant determines each 5-6 of yeast clone with identical zeocin concentration level, specific P1 ' the amino acid whose recombinant vectors transformant that screening secreting, expressing amount is the highest must change at each P1 ' that copy number is identical to compare between serial carrier transformant reaches a conclusion.
(6) liquid culture of yeast transformant and methanol induction are expressed:
P1 ' the change determining step (5) to have identical exogenous origin gene integrator copy number transforms subgroup P1 '-luciferase and the contrast of unloaded transformant is seeded to BMGY liquid nutrient medium, and 25-30 DEG C of shaking table is cultured to OD
600measured value about 2, collects yeast cell and (is diluted to OD so that BMMY liquid nutrient medium is resuspended
600value is about 1) and continue to cultivate at 25-30 DEG C of shaking table to induce external source recombination secreting, expressing, detailed methanol induction Pichiapastoris foreign gene secreting, expressing operation steps is see the EasySelectTMPichiaExpressionKit specification sheets of Invitrogen company.From start with BMMY induction (0h), receive a sample every 24h, coinduction 120h, the yeast culture matter sample collected by centrifugation supernatant of results.
(7) the high recombinant vectors transformant of expression level is screened
Detect the fluorescent signal that luciferase genetic expression produces, to determine the secreting, expressing level of luciferase gene.
Detection method: 80 μ L yeast culture supernatants are added in each hole of the real end 96 orifice plate (purchased from American PerkinElmer company) of CulturPlateTM-96 black, carry out fluorescent signal detection under the excitation wavelength of 515nm and the determined wavelength condition of 528nm.By comparing, filter out the amino acid whose recombinant vectors transformant of the highest specific P1 ' of output, result is see Fig. 5.
In addition to the above methods, also can with ELISA or Westernblotting direct-detection target protein expression amount.
From Fig. 4 and Fig. 5, different P1 ' amino-acid residues causes the secreting, expressing level being integrated into the restructuring foreign gene Luciferase of Yeast genome with secretion expression carrier pPICZ α A to produce sizable difference, the difference of copy number obviously can not change the difference of the relative expression levels that each P1 ' amino-acid residue causes, and (high is still high, low is still low), each transformed yeast strain secreting, expressing level only can be made to be similar to and to change on year-on-year basis.Compare with Fig. 4 and Fig. 5 and just can find out: which in 20 amino-acid residues can bring the highest secreting, expressing level, and this highest level can have many high (the secreting, expressing level relative to other amino-acid residues bring), different with the foreign gene difference of secreted expression.For Luciferase, l-asparagine (Asn) can show the highest secreting, expressing level when P1 ' site, is equivalent to about 4 times of the halfcystine (Cys) of minimum level.Just can pick out the highest Asn-Luciferase bacterial strain of secreting, expressing amount accordingly, in order to express this foreign protein on a large scale.
The Yeast transformant preparation method of embodiment 3 one kinds of high-level secretory expression KITligand albumen
KITligand protein preparation method described in the present embodiment is substantially identical with embodiment 1 step, unlike: with KITligand gene order for stencil design 20 forward series primers and a reverse primer.Wherein, application determination of protein concentration method (as BCA assay method), SDS-PAGE protein electrophoresis is or/and WesternBloting immunoblotting detects KITligand secreting, expressing level; The comparison of each P1 ' amino acid whose yeast transformant secreting, expressing level, see table 1.
Described 20 forward primers comprising the codon of 20 kinds of natural amino acids are respectively:
SEQIDNO.63：
Glu (E): 5 '-CTCGAGAAAAGAGAA
gAAGGTATCTGTAGAAATAGAGTC-3 ' (introduce XhoI site, underscore part is that KITligandCDS5 ' holds complementary region);
SEQIDNO.64：
Ala(A):5’-CTCGAGAAAAGAGCTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.65：
Arg(R):5’-CTCGAGAAAAGAAGAGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.66：
Asn(N):5’-CTCGAGAAAAGAAACGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.67：
Asp(D):5’-CTCGAGAAAAGAGATGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.68：
Cys(C):5’-CTCGAGAAAAGATGTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.69：
Gln(Q):5’-CTCGAGAAAAGACAAGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.70：
Gly(G):5’-CTCGAGAAAAGAGGTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.71：
His(H):5’-CTCGAGAAAAGACATGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.72：
Ile(I):5’-CTCGAGAAAAGAATTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.73：
Leu(L):5’-CTCGAGAAAAGACTTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.74：
Lys(K):5’-CTCGAGAAAAGAAAAGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.75：
Met(M):5’-CTCGAGAAAAGAATGGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.76：
Phe(F):5’-CTCGAGAAAAGATTTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.77：
Pro(P):5’-CTCGAGAAAAGACCAGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.78：
Ser(S):5’-CTCGAGAAAAGATCTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.79：
Thr(T):5’-CTCGAGAAAAGAACTGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.80：
Trp(W):5’-CTCGAGAAAAGATGGGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.81：
Tyr(Y):5’-CTCGAGAAAAGATATGAAGGTATCTGTAGAAATAGAGTC-3’；
SEQIDNO.82：
Val(V):5’-CTCGAGAAAAGAGTTGAAGGTATCTGTAGAAATAGAGTC-3’。
The described unique reverse primer matched with above-mentioned 20 forward primers is:
SEQIDNO.83:5 '-GCGGCCGCGGCGGCAACTGGAGGCAACATG-3 ' (introduce NotI site, underscore part is that KITligandCDS3 ' holds complementary region)
The Yeast transformant preparation method of embodiment 4 one kinds of high-level secretory expression FGF16 albumen
FGF16 protein preparation method described in the present embodiment is substantially identical with embodiment 1 step, unlike: with FGF16 gene order for stencil design 20 forward series primers and a reverse primer.Wherein, application determination of protein concentration method (as BCA assay method), SDS-PAGE protein electrophoresis is or/and WesternBloting immunoblotting detects FGF16 secreting, expressing level; The comparison of each P1 ' amino acid whose yeast transformant secreting, expressing level, see table 1.
Described 20 forward primers comprising the codon of 20 kinds of natural amino acids are respectively:
SEQIDNO.84：
Glu (E): 5 '-CTCGAGAAAAGAGAA
aTGGCTGAAGTTGGTGGTGTTTTC-3 '; (introduce XhoI site, underscore part is that FGF16CDS5 ' holds complementary region)
SEQIDNO.85：
Ala(A):5’-CTCGAGAAAAGAGCTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.86：
Arg(R):5’-CTCGAGAAAAGAAGAATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.87：
Asn(N):5’-CTCGAGAAAAGAAACATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.88：
Asp(D):5’-CTCGAGAAAAGAGATATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.89：
Cys(C):5’-CTCGAGAAAAGATGTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.90：
Gln(Q):5’-CTCGAGAAAAGACAAATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.91：
Gly(G):5’-CTCGAGAAAAGAGGTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.92：
His(H):5’-CTCGAGAAAAGACATATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.93：
Ile(I):5’-CTCGAGAAAAGAATTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.94：
Leu(L):5’-CTCGAGAAAAGACTTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.95：
Lys(K):5’-CTCGAGAAAAGAAAAATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.96：
Met(M):5’-CTCGAGAAAAGAATGATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.97：
Phe(F):5’-CTCGAGAAAAGATTTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.98：
Pro(P):5’-CTCGAGAAAAGACCAATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.99：
Ser(S):5’-CTCGAGAAAAGATCTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.100：
Thr(T):5’-CTCGAGAAAAGAACTATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.101：
Trp(W):5’-CTCGAGAAAAGATGGATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.102：
Tyr(Y):5’-CTCGAGAAAAGATATATGGCTGAAGTTGGTGGTGTTTTC-3’；
SEQIDNO.103：
Val(V):5’-CTCGAGAAAAGAGTTATGGCTGAAGTTGGTGGTGTTTTC-3’。
The described unique reverse primer matched with above-mentioned 20 forward primers is:
SEQIDNO.104：
5 '-GCGGCCGC
tCTGTAGTGGAACAAGTCTCTAG-3 ' (introduce NotI site, underscore part is that FGF16CDS3 ' holds complementary region)
The Yeast transformant preparation method of embodiment 5 one kinds of high-level secretory expression FGF20 albumen
FGF20 protein preparation method described in the present embodiment is substantially identical with embodiment 1 step, unlike: with FGF20 gene order for stencil design 20 forward series primers and a reverse primer.Wherein, application determination of protein concentration method (as BCA assay method), SDS-PAGE protein electrophoresis is or/and WesternBloting immunoblotting detects FGF20 secreting, expressing level; The comparison of each P1 ' amino acid whose yeast transformant secreting, expressing level, see table 1.
Described 20 forward primers comprising the codon of 20 kinds of natural amino acids are respectively:
SEQIDNO.105：
Glu (E): 5 '-CTCGAGAAAAGAGAA
aTGGCTCCATTGGCTGAAGTTGG-3 ' (introduce XhoI site, underscore part is that FGF20CDS5 ' holds complementary region);
SEQIDNO.106：
Ala(A):5’-CTCGAGAAAAGAGCTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.107：
Arg(R):5’-CTCGAGAAAAGAAGAATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.108：
Asn(N):5’-CTCGAGAAAAGAAACATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.109：
Asp(D):5’-CTCGAGAAAAGAGATATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.110：
Cys(C):5’-CTCGAGAAAAGATGTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.111：
Gln(Q):5’-CTCGAGAAAAGACAAATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.112：
Gly(G):5’-CTCGAGAAAAGAGGTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.113：
His(H):5’-CTCGAGAAAAGACATATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.114：
Ile(I):5’-CTCGAGAAAAGAATTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.115：
Leu(L):5’-CTCGAGAAAAGACTTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.116：
Lys(K):5’-CTCGAGAAAAGAAAAATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.117：
Met(M):5’-CTCGAGAAAAGAATGATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.118：
Phe(F):5’-CTCGAGAAAAGATTTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.119：
Pro(P):5’-CTCGAGAAAAGACCAATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.120：
Ser(S):5’-CTCGAGAAAAGATCTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.121：
Thr(T):5’-CTCGAGAAAAGAACTATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.122：
Trp(W):5’-CTCGAGAAAAGATGGATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.123：
Tyr(Y):5’-CTCGAGAAAAGATATATGGCTCCATTGGCTGAAGTTGG-3’；
SEQIDNO.124：
Val(V):5’-CTCGAGAAAAGAGTTATGGCTCCATTGGCTGAAGTTGG-3’。
The described unique reverse primer matched with above-mentioned 20 forward primers is:
SEQIDNO.125:5 '-GCGGCCGCAGTGTACATCAACAAGTCCTTG-3 ' (introduce NotI site, underscore part is that FGF20CDS3 ' holds complementary region)
The Yeast transformant preparation method of embodiment 6 one kinds of high-level secretory expression Irisin albumen
Irisin protein preparation method described in the present embodiment is substantially identical with embodiment 1 step, unlike: with Irisin gene order for stencil design 20 forward series primers and a reverse primer.Wherein, application determination of protein concentration method (as BCA assay method), SDS-PAGE protein electrophoresis is or/and WesternBloting immunoblotting detects Irisin secreting, expressing level; The comparison of each P1 ' amino acid whose yeast transformant secreting, expressing level, see table 1.
Described 20 forward primers comprising the codon of 20 kinds of natural amino acids are respectively:
SEQIDNO.126：
Glu (E): 5 '-CTCGAGAAAAGAGAA
tCTCCATCTGCTCCAGTTAACG-3 ' (introduce XhoI site, underscore part is that IrisinCDS5 ' holds complementary region);
SEQIDNO.127：
Ala(A):5’-CTCGAGAAAAGAGCTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.128：
Arg(R):5’-CTCGAGAAAAGAAGATCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.129：
Asn(N):5’-CTCGAGAAAAGAAACTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.130：
Asp(D):5’CTCGAGAAAAGAGATTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.131：
Cys(C):5’-CTCGAGAAAAGATGTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.132：
Gln(Q):5’-CTCGAGAAAAGACAATCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.133：
Gly(G):5’-CTCGAGAAAAGAGGTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.134：
His(H):5’-CTCGAGAAAAGACATTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.135：
Ile(I):5’-CTCGAGAAAAGAATTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.136：
Leu(L):5’-CTCGAGAAAAGACTTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.137：
Lys(K):5’-CTCGAGAAAAGAAAATCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.138：
Met(M):5’-CTCGAGAAAAGAATGTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.139：
Phe(F):5’-CTCGAGAAAAGATTTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.140：
Pro(P):5’-CTCGAGAAAAGACCATCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.141：
Ser(S):5’-CTCGAGAAAAGATCTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.142：
Thr(T):5’-CTCGAGAAAAGAACTTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.143：
Trp(W):5’-CTCGAGAAAAGATGGTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.144：
Tyr(Y):5’-CTCGAGAAAAGATATTCTCCATCTGCTCCAGTTAACG-3’；
SEQIDNO.145：
Val(V):5’-CTCGAGAAAAGAGTTTCTCCATCTGCTCCAGTTAACG-3’。
The described unique reverse primer matched with above-mentioned 20 forward primers is:
SEQIDNO.146：
5 '-GCGGCCGCTTCCTTCATAGTAACTTCGTCC-3 ' (introduce NotI site, underscore part is that IrisinCDS3 ' holds complementary region)
The comparison of each P1 ' amino acid whose yeast transformant secreting, expressing level of the recombination that table 1 yeast secreted expression is optimized
Note: * is unloaded yeast secreted expression carrier pPICZ α A negative control.
As known from Table 1: different P1 ' amino-acid residues causes the secreting, expressing level being integrated into the restructuring foreign gene of Yeast genome with secretion expression carrier pPICZ α A to produce sizable difference, which in 20 amino-acid residues can bring the highest secreting, expressing level, and this highest level can have many high (the secreting, expressing level relative to other amino-acid residues bring), different with the foreign gene difference of secreted expression.Mentioned above principle is not only applicable to the reporter gene as Venus, Luciferase, applicable equally for common recombination, utilizes this principle just can pick out the highest yeast strain of secreting, expressing amount, in order to express foreign protein on a large scale.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (3)
1. a preparation method for the yeast transformant of secreting, expressing foreign protein, is characterized in that, comprises the following steps:
(1) primer of cloned foreign gene CDS is designed: 5 ' end of forward primer introduces XhoI site, and the codon of 20 kinds of natural amino acids is introduced respectively in the P1 ' site corresponding to Kex2 proteolytic enzyme identified region, 5 ' end of reverse primer introduces NotI site, obtains 20 to serial primer; Described P1 ' site is first amino acid of Kex2 shearing site C-end direction;
(2) TA clones interested foreign gene CDS: with containing (1) described 20 in steps to cDNA or DNA of the complete CDS sequence of the interested target gene of serial primer for template, complete CDS gene fragment is gone out with the described serial primer amplification of step (1), reclaim PCR primer TA and connect into TA cloning vector pMD20-T, obtain Series P 1 ' and change carrier group P1 '-CDS-pMD20-T, identical with design through sequence verification all carriers P1 ' site amino acids codon, and foreign gene CDS is defined as correct expressible nucleotide sequence;
(3) sub clone construction yeast carrier for expression of eukaryon group: by carrier group the P1 '-CDS-pMD20-T of step (2) gained and Yeast expression carrier pPICZ α A, double digested and reclaim through XhoI and NotI respectively, connect reagent with SolutionI to connect, obtain Series P 1 ' and change carrier group P1 '-CDS-pPICZ α A;
(4) recombinant vectors group is transformed in yeast host: by the Series P 1 ' of step (3) gained change carrier group P1 '-CDS-pPICZ α A after the linearizing of SacI single endonuclease digestion, reclaim digestion products, proceed in the X-33 Host Strains of Pichia pastoris through lithium chloride conversion method again, obtain positive transformant through zeocin90-110 μ g/ml screening;
(5) yeast transformant copy number is determined: the YPD be transferred to by the positive transformant of step (4) gained containing zeocin increasing concen-trations is dull and stereotyped, allly can not be considered as the yeast transformant with identical exogenous origin gene integrator copy number at the yeast transformant of next greater concn horizontal growth at the YPD grow on plates of same zeocin concentration level, obtain conversion subgroup the P1 '-CDS with identical exogenous origin gene integrator copy number thus;
(6) liquid culture of yeast transformant and methanol induction are expressed: have conversion subgroup the P1 '-CDS of identical exogenous origin gene integrator copy number and the unloaded transformant of step (5) gained are seeded to BMGY substratum, and 25-30 DEG C of shaking table is cultured to OD
600for 1.8-2.2, collect yeast cell BMMY substratum resuspended, be diluted to OD
600for 0.9-1.1, methanol induction 110-130h is centrifugal and collect supernatant;
(7) the highest recombinant vectors transformant of expression level is screened: by foreign recombinant proteins output in the supernatant of analytical procedure (6) gained, specific P1 ' the amino acid whose recombinant vectors transformant that screening secreting, expressing amount is the highest must change at each P1 ' that copy number is identical between serial carrier transformant P1 '-CDS and compares
Obtain the amino acid whose recombinant vectors transformant of the highest P1 ' of expression level;
The forward primer of the codon of the described 20 kinds of natural amino acids of step (1) is respectively:
SEQIDNO.1：
Glu：5’-CTCGAGAAAAGAGAA-CDS-3’；
SEQIDNO.2：
Ala：5’-CTCGAGAAAAGAGCT-CDS-3’；
SEQIDNO.3：
Arg：5’-CTCGAGAAAAGAAGA-CDS-3’；
SEQIDNO.4：
Asn：5’-CTCGAGAAAAGAAAC-CDS-3’；
SEQIDNO.5：
Asp：5’-CTCGAGAAAAGAGAT-CDS-3’；
SEQIDNO.6：
Cys：5’-CTCGAGAAAAGATGT-CDS-3’；
SEQIDNO.7：
Gln：5’-CTCGAGAAAAGACAA-CDS-3’；
SEQIDNO.8：
Gly：5’-CTCGAGAAAAGAGGT-CDS-3’；
SEQIDNO.9：
His：5’-CTCGAGAAAAGACAT-CDS-3’；
SEQIDNO.10：
Ile：5’-CTCGAGAAAAGAATT-CDS-3’；
SEQIDNO.11：
Leu：5’-CTCGAGAAAAGACTT-CDS-3’；
SEQIDNO.12：
Lys：5’-CTCGAGAAAAGAAAA-CDS-3’；
SEQIDNO.13：
Met：5’-CTCGAGAAAAGAATG-CDS-3’；
SEQIDNO.14：
Phe：5’-CTCGAGAAAAGATTT-CDS-3’；
SEQIDNO.15：
Pro：5’-CTCGAGAAAAGACCA-CDS-3’；
SEQIDNO.16：
Ser：5’-CTCGAGAAAAGATCT-CDS-3’；
SEQIDNO.17：
Thr：5’-CTCGAGAAAAGAACT-CDS-3’；
SEQIDNO.18：
Trp：5’-CTCGAGAAAAGATGG-CDS-3’；
SEQIDNO.19：
Tyr：5’-CTCGAGAAAAGATAT-CDS-3’；
SEQIDNO.20：
Val：5’-CTCGAGAAAAGAGTT-CDS-3’。
2. the preparation method of the yeast transformant of secreting, expressing foreign protein according to claim 1, is characterized in that, the concentration of zeocin described in step (4) is 100 μ g/ml.
3. the preparation method of the yeast transformant of secreting, expressing foreign protein according to claim 1, is characterized in that, the concentration of the zeocin of YPD flat board described in step (5) is followed successively by 200 μ g/ml, 500 μ g/ml and 1000 μ g/ml.
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| Systematic Single-Cell Analysis of Pichia pastoris Reveals Secretory Capacity Limits Productivity;Kerry Routenberg Love et al;《PLoS ONE》;20120607;第7卷(第6期);e37915 * |
| The specificity of the neuroendocrine convertase PC3 is determined by residues NH2‐ and COOH‐terminal to the cleavage site;Elizabeth C. Ledgerwood et al;《BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL》;19960830;第39卷(第6期);摘要、第1173页第1段、表1 * |
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