CN102660574A - Method for establishing a system of dual-promoter methanol yeast efficiently expressing recombinant human metallothionein, and preparation and purification method of the protein - Google Patents
Method for establishing a system of dual-promoter methanol yeast efficiently expressing recombinant human metallothionein, and preparation and purification method of the protein Download PDFInfo
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Abstract
The invention discloses a method which is feasible in pharmaceutical industry for efficient secretion expressing, preparing and purifying of gene recombinant human metallothionein or analogues thereof. The method is characterized by introducing exogenous genes containing dual-promoters into relevant host yeast cells in transformation or double crossover recombination manner by using plasmid vector pPICZ alpha-MT of exogenous metal sulfur protein genes which contain methanol response element AOX and contain or not contain metal response element MRE to construction expression engineering cells containing the dual-promoters in the methanol yeast host cells. According to the invention, several problems with traditional preparation method of metallothionein, such as high production cost, slaughter and organ harvesting to large number of animals, virus pollution derived from animal, endotoxin pollution derived from bacterial, immunogen of non-humanized protein and the like, are avoided effectively,.
Description
Technical field
The present invention relates to a kind of method that the double-promoter methanol yeast that embeds methyl alcohol response element and metal response element efficiently expresses recombination human metal thioalbumen (rhMT) system of setting up; And the technology of utilizing this system expression, preparation, this target protein-rhMT of purifying; Belong to field of biological pharmacy; Its key is with a kind of methyl alcohol response element AOX that contains; And contain or the vector plasmid pPICZ α-MT of the external source metallothionein gene of containing metal response element MRE not; Through calcium phosphate transfection (transformation), electric shock transfection or homologous recombination modes such as (double crossover recombination), this foreign gene that contains double-promoter is introduced relevant host's yeast cell, its objective is the feasibility embodiment that a kind of suitable large-scale industrialization low cost prodn human metal thioalbumen is provided.
Background technology
Nineteen fifty-seven; U.S. scientist Margoshes at the research metal in biologically time spent of doing; From the horse kidney, find I-type and II-type cadmium metal sulfoprotein (Margoshes M., Vallee B.L.A cadmium protein from equine kidney cortex.J.Ame.Chem.Soc.79:4813-4814 (1957)) first.RhMT is one type and is rich in the halfcystine group; Various heavy there is high affinity; Under certain potential of hydrogen (pH value) and cellular oxidation-reduction potential environment (but mat organoid somewhere gsh has reduced form and oxidized form ratio, the demonstration of GSH/GSSG ratio) condition, be prone to and metals ion bonded polypeptide proteins such as a plurality of cadmiums, zinc.Mammiferous rhMT is made up of about 61 amino acid, contains 20 halfcystines and 7 metals ions.RhMT is as a kind of stress protein, Green Tea Extract oxygenant, cell zinc ion mediation agent etc., has storage, transportation and the metabolism, lead discharging zinc supplementation, the radioprotective that improve body immunity, opposing heavy metal poisoning, participate in trace element, delays senility, protects the effective biological function of multiple uniqueness such as each organ of human body cardiopulmonary.
Generally lack a kind of especially feasible production preparation and purification process of pharmaceutical industry of people source rhMT or its analogue of secretory gene recombinant mammalian that efficiently express up to now.Fudan University discloses a kind of preparation method of rhMT on patent CN01131966.6; But belong to monkey but not human metal thioalbumen; And need the excision of zymoplasm on the post to be used for the gsh GST segment of affinity chromatography, cost is too high, and the industrial production of not having is worth; Peking University discloses a kind of selection fermentation and extraction process of brewing yeast metallothionein producing strain on patent CN96100897.0, induce preparation Cu-MT with mantoquita; Ji'nan University discloses a kind of expression method of inducing escherichia coli expression soluble fusion protein brewing yeast metallothionein mature peptide CBD-intrin1-MT with IPTG on patent CN201110028345.4, utilized intrin1 pulsating from shearing function well; But these two patents relate to non-human metal thioalbumen; Ji'nan University discloses to produce on patent CN200610112960.2 and has utilized small molecules ubiquitin relevant modifications factor mature peptide (Small Ubiquitin-related Modifier; SUMO) carry out the method for amalgamation and expression production people I shaped metal sulfoprotein hMT-I; Make this fusion segment can be by ubiquitin relevant modifications factor protein enzyme 1 (Ubiquitin-like protease 1) hydrolysis in the prokaryotic organism body; With release soluble hMT-I; But belong in prokaryotic organism and to use expensive IPTG to induce down to express, have the bacterial endotoxin Pollution risk when target protein is used for medicine so this is stated; Other has one kind of patent CN200910246446.1 human metal thioalbumen to be received the method for fusion rotein of production Urogastrone and rhMT of Urogastrone's C end; Only be used for preparing the treatment burn, or scald or the application and the application in preparation skin care medicine of the medicine of wound or ulcer, do not have ubiquity; U.S. Pat 5824512 has been described a kind of expression Mal-MT Expression of Fusion Protein, and its purposes is merely the environmental protection waste treatment.In sum; A kind ofly be fit to generally that pharmaceutical heavy industrialization is expressed cheaply, the industrialization technology of preparation, purifying, detection human metal thioalbumen presses for exploitation, especially considers the bright prospects of people source rhMT hMT-3 on the treatment nerve degenerative diseases.HMT-3 mainly is distributed in the star property spongiocyte of cns, concentrates to be present in cell paste and the projection, and next is present in the neuronal cell.Through discovering of mouse thorn wound model and ischemia model, hMT-3 participates in the central nervous system injury reparation.A kind of mechanism of Parkinson's disease (PD) is owing to the factors such as 6 monohydroxy Dopamine HCLs induce radical to produce; And some rhMT isomer inductor of hMT-3 especially in the brain; Can prevent this neuron excitotoxicity like oxidative pressure, cytokine and inflammatory process etc., this is relevant with rhMT removing radical.
Developing into of Protocols in Molecular Biology utilizes bio-reactor production foreign protein that many ways and meanses are provided.Up to the present, developed multiple protein expression systems such as intestinal bacteria, yeast, insect, mammalian cell, transgenic plant, wherein the yeast cell to express system is the novel expression system that latest developments are got up.What use at first is yeast saccharomyces cerevisiae.Hitzeman in 1981 etc. have successfully expressed human interferon with yeast saccharomyces cerevisiae, but this system has certain limitation, as lack strong promotor, and secernment efficiency is poor, plasmid instability etc.Therefore, people have sought new yeast expression system---pichia spp, i.e. methyl alcohol nutritional type expression system on this basis again.This expression system is widely used at present.This system not only has high expression level, high stable, high excretory characteristics, and its host's methanol yeast-pichia pastoris phaff (Picchia pastoris) has the characteristic of high-density growth.Methanol yeast is the yeast of methyl alcohol capable of using as sole carbon source; Mainly contain three kinds of H.Polymorpha, Candida Bodinii, Pichia Pastoris, wherein pichia spp (Pichia Pastoris) makes with the most use, the most extensive as gene expression system.Compare with gene expression system in the past, it has unapproachable high expression level characteristic, has been considered to one of proteinic instrument of production that has most development prospect.Therefore the research of this expression system is in recent years developed rapidly, has expressed multiple foreign protein with commercial value therein, comprises human protein HuMOR, hTopoI, hGM-CSF, SAP, C1-Inh, SolRII.As far back as nineteen ninety-five, existing more than 40 kind of foreign protein obtains to express in this host bacterium.And recent years, the annual expressed exogenous gene of reporting in pichia spp just had tens kinds, and many year by year.The widespread use of Bichi yeast system; Reason be this system except have that general yeast has with intestinal bacteria than low toxicity or nontoxic, cultivate than the characteristics such as production cost is low with mammalian cell CHO, also have and contain a lot of distinctive advantages: contain at present the strongest promotor-AOX (alcohol oxidase gene), expression level high, be prone to secreting, expressing, translation back be prone to processing treatment, high-density culture, zymotechnique ripe, be prone to amplify, product is easily separated, foreign protein genes inheritance stability etc.
Promotor (promoter) is meant on the dna molecular by RNA polymerase identification and combines to form the zone of initial transcription complex, and it comprises that also some regulate the binding site of protein factors.As the binding site of promotor with its upper reaches transcriptional regulation protein at a distance is included in together, be called promoter region (promoter region).Promotor is extremely important for Recombinant Protein Expression.An eukaryotic gene has a plurality of regulating and controlling sequences usually, mainly is present in promoter region.The promotor that recombinant protein is efficiently expressed has usually can start transcription of foreign genes and two characteristics of regulation and control abduction delivering effectively.A plurality of metal response elements and metalloid response element are contained in metal hydrosulfide protein gene promoter district, metal response element (MRE) and metalloid response element (MLS, MRE-like sequence).What is interesting is that in the metal regulatory gene, it is position and the direction that does not rely on MRE that heavy metal is reacted, but need the copy more than 2 at least.The heavy metal that the antagonism of metal hydrosulfide protein protection cell is too much, this gene is only expressed in basic horizontal in the ordinary course of things, but next with high level expression inducing of heavy metal ion.A plurality of MRE sequences in metal hydrosulfide protein gene promoter district are undertaken the inducibility of metal are replied effect.
Summary of the invention
Main innovation of the present invention is with containing methyl alcohol response element AOX1; And contain or the plasmid of the external source metallothionein gene of containing metal response element MRE not; Through the mode of transfection (transformation) or homologous recombination (double crossover recombination), this foreign gene that contains double-promoter is introduced relevant host's yeast cell.Like this, inserting under the situation of the gene segment of promotor of containing metal sulfoprotein own such as MRE etc. not, the external source metallothionein gene is only by methyl alcohol response element AOX1 regulatory transcription; When what insert is that the external source metallothionein gene will be by methyl alcohol response element AOX1 and promotor of rhMT own such as the common regulatory transcription of metal response element under the situation of promotor of containing metal sulfoprotein own such as MRE etc.Compare with single promotor, double-promoter has strengthened the expression efficiency of rhMT and the handiness of regulation and control significantly.
More particularly, the invention provides a kind of double-promoter methanol yeast of setting up and efficiently express the method for recombination human metal thioalbumen system and the preparation purification technique of this target protein.
Further, said target protein is human metal thioalbumen (hMT), comprise the human metal thioalbumen isomer (hMT-1, hMT-2, hMT-3, hMT-4) and analogue, segment.
Further, described human metal thioalbumen isomer (hMT-3 hMT-4) has following protein sequence for hMT-1, hMT-2:
SEQ ID NO.1, I-type human metal thioalbumen (metallothionein-1, hMT-1):
1?mdpncscatg?gsctctgsck?ckeckctsck?ksccsccpms?cakcaqgcic?kgasekcscc61a
SEQ ID NO 2, II-type human metal thioalbumen (metallothionein-2, hMT-2):
1?mdpncgcaag?dsctcagsck?ckeckctsck?ksccsccpvg?cakcaqgcic?kgasdkcscc61a
SEQ ID NO.3, III-type human metal thioalbumen (metallothionein-3, hMT-3):
1?mdpetcpcps?ggsctcadsc?kcegckctsc?kksccsccpa?ecekcakdcv?ckggeaaeae61?aekcsccq
SEQ ID NO 4.IV-type human metal thioalbumen (metallothionein-4, hMT-4):
1?mdprecvcms?ggicmcgdnc?kcttcncktc?rksccpccpp?gcakcargci?ckggsdkcsc61?cp
Further; For obtaining said target gene sequences, contain the promoter regulation sequence, secreting the targeting signal expressed sequence; And the method for introducing suitable restriction endonuclease site; To make the described double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system that contains, may further comprise the steps:
A) employing genetic engineering technique or the directly method of DNA chemosynthesis; Introduce one or more restriction endonuclease (XhoI, Cla I, Pst I, EcoR I, Pml I, Sfi I, Bsmb I, Asp718 I, Kpn I, Xho I, Sac II, Not I, Xba I) restriction enzyme site site that the termination meets the pPICZ of vector plasmid shown in accompanying drawing α and insert and require, and contain the ripe gene segment of the pairing DNA of aminoacid sequence of coding claim 2, a kind of human metal thioalbumen described in 3 and/or contain the pulsating dna double spiral long-chain of its promoter DNA (MT gene segment).The genetic engineering technique that is adopted includes but not limited to the gene clone method that those skilled in the art know; For example; From the full RNA of human body tissue extraction, design PCR primer, carry out the RT-PCR reaction obtain cDNA, gene sequencing, sepharose chromatogram detect confirm, site-directed point mutation, or the clone of dna segment or plasmid, enzyme cut, connect, extraction, purifying, pcr amplification, conversion etc.
B) use dna ligase; The MT gene segment is inserted respective limits property endonuclease restriction enzyme site on the vector plasmid pPICZ α; Make the MT gene segment and the reorganization methanol yeast secretion expression system of alcohol oxidase (AOX1) gene promoter of the 5 ' control region (including promoter element) that contains a kind of pichia pastoris phaff bacterium source integrate (chimeric), carrier construction plasmid pPICZ α-MT.
C) will go up the constructed last goal gene segment of vector plasmid pPICZ α-MT of a step; Comprise regulator sequences such as guiding peptide expressed sequence, target protein matter encoding sox segment, promotor; Ordinary method through transfection (transformation) or homologous recombination (double crossover recombination); Preferred homologous recombination method; This foreign gene that contains double-promoter is introduced relevant host's yeast cell, made up and contain double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system.
Further, constructed vector plasmid pPICZ α-MT contains the genetic expression structural formula: 5 '--S-T-3 ', and in the formula: S is guiding peptide expressed sequence; Its encoding amino acid sequence is EAEAEFR; Or EAEFR, or EFR, or the α mating factor gene leader peptide sequence in yeast saccharomyces cerevisiae source (is an AMF prepro sequence; Its sequence is 85 amino acid whose gene fragments of a kind of coding, contains two codons of yeast KEX-2 proteolytic enzyme processing site Lys-Arg); T is the ripe gene segment of the pairing DNA of aminoacid sequence of coding claim 2, a kind of human metal thioalbumen described in 3, and this segment contains or not containing metal response element (MRE) and/or metalloid response element (MLS, MRE-like sequence).
Further, described methanol yeast is Pichia (Pichia) or Hansenula (Hansenula) or torulopsis (Torulopsis), preferred Pichia; Adopting the host bacterium is GS115, GS190, GS200, JC220, JC227, JC254, JC304, JC305, or SMD16, SMD1163, SMD1165, SMD1168, or X33, or KM71, preferred GS115.
The present invention also studies the preparation purification technique of sulphur target protein recombination human metal thioalbumen; Promptly adopt the optimization for fermentation technology technology; Comprise the rhMT secreting, expressing engineering strain of selecting to contain double-promoter; Through being fit to grown cultures and expressing cultivation, obtain the fermented liquid of secreting, expressing target protein; The fermented liquid that further will contain target protein is through the optimized proteins purification technique, obtain purity greater than 60% bullion or purity greater than 90% elaboration or purity greater than 99% pure article.
Further; Said zymotechnique technology comprises: 1). utilization fermentating metabolism stream, energy stream, oxygen TRANSFER MODEL analysis software or simulation software or single factor experiment design or orthogonal experimental design etc. are optimized processing parameters such as the fermention medium selection, pH value, temperature of the inoculum size that influence saccharomycetes to make fermentation, inoculation time, dissolved oxygen amount, the most suitable recombinant bacterial strain protein expression, with the expression amount of raising fermentation efficiency and target protein; 2). fermentation parameter is: leavening temperature is 15-30 ℃, preferred 28-30 ℃; The pH value is 3-7, preferred pH=4-6; Dissolved oxygen amount DO is greater than 20%, and preferred 30%; Glucose or glycerine exhaust the restricted stream in back and add 2-30 hour, and preferred 12-15 hour, restricted again stream added methyl alcohol, up to fermentation ends; The whole process temperature that glucose or glycerine are depleted to fermentation ends is controlled at 15-30 ℃, preferred 19-23 ℃; The pH value is controlled at 3-7, preferred pH=4-6.
Further; Said protein purification technology; Comprise use metal chelate chromatography post (Metal Chelating Chromatography), ultrafiltration, molecular weight cutting film (Molecular Weight Cut-Off Membrane), enzyme is cut or at least a or more than one combinations of methods such as chemical chop, reversed-phase liquid chromatography (Reverse Phase HPLC), cationic exchange coloum, macroporous resin column, hydrophobic chromatography separator column, dialysis, spinning, purifying is prepared rhMT that purity meets the requirements as medicine or other purposes.
Effect effect of the present invention is:
A kind of especially feasible production preparation and purification process of pharmaceutical industry of people source rhMT or its analogue of secretory gene recombinant mammalian that efficiently express is provided; It is characterized by; Through with a kind of methyl alcohol response element AOX1 that contains; And contain or the vector plasmid pPICZ α-MT of the external source metallothionein gene of containing metal response element MRE not; Through the mode of transfection (transformation) or homologous recombination (double crossover recombination), this foreign gene that contains double-promoter is introduced relevant host's yeast cell, made up the methanol yeast host gene engineering bacteria that contains double starting; Utilization fermentating metabolism stream, energy stream, oxygen TRANSFER MODEL analysis software or simulation software or single factor experiment design or orthogonal experimental design etc. are optimized the optimization of technological parameter such as the fermention medium selection, pH value, temperature of the inoculum size that influences saccharomycetes to make fermentation, inoculation time, dissolved oxygen amount, the most suitable recombinant bacterial strain protein expression; Significantly improve fermentation efficiency, the expression amount of purpose product is significantly improved; Prepare purifying process through optimizing target protein matter; Comprise use metal chelate chromatography post (Metal Chelating Chromatography), ultrafiltration, molecular weight cutting film (Molecular Weight Cut-Off Membrane), enzyme is cut or at least a or more than one combinations of methods such as chemical chop, reversed-phase liquid chromatography (Reverse Phase HPLC), cationic exchange coloum, macroporous resin column, hydrophobic chromatography separator column, dialysis, spinning, purifying is prepared rhMT that purity meets the requirements as medicine or other purposes.The present invention has avoided among the traditional metal sulfoprotein preparation method production cost high effectively, and animal slaughtered with organ in a large number win, problems such as animal source virus pollution, bacterial origin contaminated with endotoxins, the proteic immunogen of non-humanization.
Description of drawings
Accompanying drawing is double-promoter methanol yeast efficient expression vector plasmid figure.
Embodiment
Embodiment one:
Select GS115 methanol yeast list bacterium colony through the dull and stereotyped band double-promoter secreting, expressing recombination II-type human metal thioalbumen of cultivating (rhMT-2) of yeast extract powder peptone dextrose culture-medium (YEPD); The bottle concussion of shaking placing 30 ℃ of shaking tables is cultivated 12-24 hour to OD600nm=0.20-0.60; After transfer to 1.5L and shake bottle; Continue overnight cultures, carry out feed supplement, batch fermentation with the inoculum size of 5-10% then.(B.Bran, working volume Germany) is 30L to fermentor tank, adds 15L basis salt culture medium, trace element solution and certain carbon source.The fermentation of glycerine phase is omnidistance to make oxyty maintain 30% through regulating stirring velocity (500-900rpm); Dissolved oxygen electrode is INPRO6100/120/T/N (Metler-Toledo; Switzerland), add ammoniacal liquor, control pH=6.5 through stream; PH electrode is 405-DPAS-SC-K8S/120 (Metler-Toledo, a Switzerland).When OD600nm=0.30-0.50, get into the methanol induction expression phase, in 2 hours, methyl alcohol is added to 0.2%.When methyl alcohol begins to be utilized, its concentration is progressively brought up to 0.5-1%, and progressively reduce culture temperature to 19-25 ℃, pH transfers to 6.8-7.0, uses or do not use metal (Zn, Cu etc.) inductor the same period.It is 0.04-0.06MPa that fermenting process is kept pressure tank.Glycerine, methyl alcohol, ammoniacal liquor all flow with the good peristaltic pump (Watson-Marlow101U/R, Britain) of correction and add.When OD600nm=0.60, when sulphydryl activity reaches 0.08, carry out blowing, feed supplement.After blowing, the feed supplement, about OD600nm=0.30-0.35, the substratum charge is about 15L, carries out cultured continuously then, continues to induce 200-300 hour.Survey target protein matter content with RP-HPLC (Agilent, the U.S.).When target protein concentration (greater than 110mg/L) in the fermented liquid when no longer increasing; Merge aforementioned blowing liquid; Prepare purifying process through aforementioned optimization target protein matter; Contain steps such as micro-filtration degerming, hydrophobic chromatography etc., obtain the recombination human metal thioalbumen rhMT-2 ultimate production of purity more than 95% greater than 2.5 grams.
Embodiment two:
The following stated equipment, instrument, instrument, detection method are like having occurred at embodiment one, and then both are identical.
Select GS115 methanol yeast list bacterium colony through the dull and stereotyped band double-promoter secreting, expressing recombination I-type human metal thioalbumen rhMT-1 that cultivates of YEPD; The bottle concussion of shaking placing 30 ℃ of shaking tables is cultivated 12-24 hour to OD600nm=0.20-0.60; After transfer to and contain the shaking in the bottle of 1.5L yeast nitrogen basic medium and seed culture medium, in 28-30 ℃ of fermentation culture 24 hours.Transferring to the 30L fermentor tank with the inoculum size of 5-10% then cultivates.In this fermentor tank, add the 13.5L salt culture medium, this substratum contains 27mlH3PO4 for every liter, 0.9g CaSO4.2H2O, 18g K2SO3,15g MgSO4.7H2O, 4.13g KOH, 40g glycerine, 4.4ml trace mineral solution.During the fermentation, use the ammoniacal liquor adjust pH, make it maintain 5.0-5.5.In 30 ℃ of logical oxygen, stirring, stirring velocity is 900-1000rpm, and adds proper quantity of defoaming agent, and oxyty is maintained more than 30%.Ferment after 24 hours, add the glycerine of the trace mineral solution that contains 12ml/L, oxyty continues to maintain more than 30%.Continue to cultivate 10 hours.When carbon source after hungry 30 minutes, add methyl alcohol, abduction delivering.The speed that has just begun to add in 5 hours methyl alcohol (100% methyl alcohol contains the trace mineral solution of 12ml/L) is low, and oxygen deprivation, so that yeast adapts to methyl alcohol.The back adds more methyl alcohol, and progressively reduces culture temperature to 19-25 ℃, and pH transfers to 6.8-7.0, is used certain density metal (Zn, Cu etc.) inductor.It is 0.04-0.06MPa that fermenting process is kept pressure tank.Glycerine, methyl alcohol, ammoniacal liquor all flow with the good peristaltic pump of correction and add.When OD600nm=0.60, when sulphydryl activity reaches 0.08, carry out blowing, feed supplement.After blowing, the feed supplement, about OD600nm=0.30-0.35, the substratum charge is about 15L, carries out cultured continuously then, continues to induce 200-300 hour.Survey target protein matter content with RP-HPLC.When target protein concentration (greater than 90mg/L) in the fermented liquid when no longer increasing; Merge and collect fermented liquid; Prepare purifying process through aforementioned optimization target protein matter; Contain that micro-filtration degerming, hydrophobic chromatography, 4 ℃ of 10000rpm are centrifugal, metal chelating column affinity chromatography etc., obtain the rhMT rhMT-1 ultimate production of purity more than 95% greater than 2.0 grams.
The protein sequence table
SEQ ID NO.1, I-type human metal thioalbumen (metallothionein-1, hMT-1):
1?mdpncscatg?gsctctgsck?ckeckctsck?ksccsccpms?cakcaqgcic?kgasekcscc61?a
SEQ ID NO 2, II-type human metal thioalbumen (metallothionein-2, hMT-2):
1?mdpncgcaag?dsctcagsck?ckeckctsck?ksccsccpvg?cakcaqgcic?kgasdkcscc61?a
SEQ ID NO.3, III-type human metal thioalbumen (metallothionein-3, hMT-3):
1?mdpetcpcps?ggsctcadsc?kcegckctsc?kksccsccpa?ecekcakdcv?ckggeaaeae61?aekcsccq
SEQ ID NO 4.IV-type human metal thioalbumen (metallothionein-4, hMT-4):
1?mdprecvcms?ggicmcgdnc?kcttcncktc?rksccpccpp?gcakcargci?ckggsdkcsc61?cp
Claims (9)
1. a foundation contains the method for double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system and the preparation purification technique of this target protein.
2. target protein as claimed in claim 1 is characterized in that, said target protein is human metal thioalbumen (hMT), comprise the human metal thioalbumen isomer (hMT-1, hMT-2, hMT-3, hMT-4) and analogue or segment.
3. human metal thioalbumen isomer as claimed in claim 2 is characterized in that, described human metal thioalbumen isomer has following protein sequence:
SEQ ID NO.1, I-type human metal thioalbumen (metallothionein-1, hMT-1):
1?mdpncscatg?gsctctgsck?ckeckctsck?ksccsccpms?cakcaqgcic?kgasekcscc61?a
SEQ ID NO 2, II-type human metal thioalbumen (metallothionein-2, hMT-2):
1?mdpncgcaag?dsctcagsck?ckeckctsck?ksccsccpvg?cakcaqgcic?kgasdkcscc61?a
SEQ ID NO.3, III-type human metal thioalbumen (metallothionein-3, hMT-3):
1?mdpetcpcps?ggsctcadsc?kcegckctsc?kksccsccpa?ecekcakdcv?ckggeaaeae61?aekcsccq
SEQ ID NO 4.IV-type human metal thioalbumen (metallothionein-4, hMT-4):
1?mdprecvcms?ggicmcgdnc?kcttcncktc?rksccpccpp?gcakcargci?ckggsdkcsc61?cp。
4. the double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system that contains as claimed in claim 1; It is characterized in that, make the described double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system that contains and may further comprise the steps:
1) employing genetic engineering technique or the directly method of DNA chemosynthesis; Introduce one or more restriction endonuclease (XhoI, Cla I, Pst I, EcoR I, Pml I, Sfi I, Bsmb I, Asp718 I, Kpn I, Xho I, Sac II, Not I, Xba I) restriction enzyme site site that the termination meets the pPICZ of vector plasmid shown in accompanying drawing α and insert and require, and contain the ripe gene segment of the pairing DNA of aminoacid sequence of coding claim 2, a kind of human metal thioalbumen described in 3 and/or contain the pulsating dna double spiral long-chain of its promoter DNA (MT gene segment).
2) use dna ligase; The MT gene segment is inserted respective limits property endonuclease restriction enzyme site on the vector plasmid pPICZ α; Make the MT gene segment and the reorganization methanol yeast secretion expression system of alcohol oxidase (AOX1) gene promoter of the 5 ' control region (including promoter element) that contains a kind of pichia pastoris phaff bacterium source integrate (chimeric), carrier construction plasmid pPICZ α-MT.
3) will go up the constructed last goal gene segment of vector plasmid pPICZ α-MT of a step; Comprise regulator sequences such as guiding peptide expressed sequence, target protein matter encoding sox segment, promotor; Ordinary method through transfection (transformation) or homologous recombination (double crossover recombination); Preferred homologous recombination method; This foreign gene that contains double-promoter is introduced relevant host's yeast cell, made up and contain double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system.
5. vector plasmid pPICZ α-MT as claimed in claim 4 and contain double-promoter methanol yeast efficient secretory expression recombination human metal thioalbumen system; It is characterized in that, contain the genetic expression structural formula: 5 '--S-T-3 ', in the formula: S is guiding peptide expressed sequence; Its encoding amino acid sequence is EAEAEFR; Or EAEFR, or EFR, or the α mating factor gene leader peptide sequence in yeast saccharomyces cerevisiae source (is an AMF prepro sequence; Its sequence is 85 amino acid whose gene fragments of a kind of coding, contains two codons of yeast KEX-2 proteolytic enzyme processing site Lys-Arg); T is the ripe gene segment of the pairing DNA of aminoacid sequence of coding claim 2, a kind of human metal thioalbumen described in 3, and this segment contains or not containing metal response element (MRE) and/or metalloid response element (MLS, MRE-like sequence).
6. like claim 1,4,5 each described methanol yeasts, it is characterized in that 1). said methanol yeast is Pichia (Pichia) or Hansenula (Hansenula) or torulopsis (Torulopsis), preferred Pichia; 2). adopting the host bacterium is GS115, GS190, GS200, JC220, JC227, JC254, JC304, JC305, or SMD16, SMD1163, SMD1165, SMD1168, or X33, preferred GS115.
7. the preparation purification technique of target protein recombination human metal thioalbumen according to claim 1; It is characterized in that; Employing optimization for fermentation technology technology; Comprise the rhMT secreting, expressing engineering strain of selecting to contain double-promoter, cultivate with expression, obtain the fermented liquid of secreting, expressing target protein as claimed in claim 2 through being fit to grown cultures; The fermented liquid that contains target protein is through the optimized proteins purification technique, obtain purity greater than 60% bullion or purity greater than 90% elaboration or purity greater than 99% pure article.
8. like the said optimization for fermentation technology technology of claim 7; It is characterized in that; The step of optimization for fermentation technology technology comprises: 1). utilization fermentating metabolism stream, energy stream, oxygen TRANSFER MODEL analysis software or simulation software or single factor experiment design or orthogonal experimental design etc. are optimized processing parameters such as the fermention medium selection, pH value, temperature of the inoculum size that influence saccharomycetes to make fermentation, inoculation time, dissolved oxygen amount, the most suitable recombinant bacterial strain protein expression, with the expression amount of raising fermentation efficiency and target protein; 2). fermentation parameter is: leavening temperature is 15-30 ℃, preferred 28-30 ℃; The pH value is 3-7, preferred pH=4-6; Dissolved oxygen amount DO is greater than 20%, and preferred 30%; Glucose or glycerine exhaust the restricted stream in back and add 2-30 hour, and preferred 12-15 hour, restricted again stream added methyl alcohol, up to fermentation ends; The whole process temperature that glucose or glycerine are depleted to fermentation ends is controlled at 15-30 ℃, preferred 19-23 ℃; The pH value is controlled at 3-7, preferred pH=4-6.
9. like the said protein purification technology of claim 7; It is characterized in that; Comprise use metal chelate chromatography post (Metal Chelating Chromatography), ultrafiltration, molecular weight cutting film (Molecular Weight Cut-Off Membrane), enzyme is cut or at least a or more than one combinations of methods such as chemical chop, reversed-phase liquid chromatography (Reverse Phase HPLC), cationic exchange coloum, macroporous resin column, hydrophobic chromatography separator column, dialysis, spinning, purifying is prepared rhMT that purity meets the requirements as medicine or other purposes.
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| CN111534534A (en) * | 2019-06-27 | 2020-08-14 | 河南大学 | Method for producing high-selenium-content protein by constructing yeast fermentation by using exogenous metallothionein |
| CN113215194A (en) * | 2021-04-13 | 2021-08-06 | 重庆绵凯生物技术研究院有限公司 | Preparation and application of fluorescent protein-transferring gene medaka for monitoring heavy metals in seawater |
| CN113215194B (en) * | 2021-04-13 | 2023-05-16 | 重庆绵凯生物技术研究院有限公司 | Preparation and application of fluorescence-transferred protein gene-modified medaka for monitoring seawater heavy metals |
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