CN1088106C - Gene engineering process of preparing spinach chloroplast triose phosphate isomerase - Google Patents

Abstract

The present invention relates to a gene engineering method of preparing spinach chloroplast triose phosphate isomerase. After spinach chloroplast triose phosphate isomer enzyme TPI genes are obtained by an oligonucleotide primer which is designed by synthesis and reverse transcription polymerase chain reaction RT-PCR, the spinach chloroplast triose phosphate isomer enzyme TPI genes are cloned into a plasmid and are controlled by a specific sequence of a protein biosynthesis initiation region of a gene expression regulation sequence of a computer-aided design, the expression of destination protein TPI in escherichia coli can maximally reach 36% of the total protein of the thallus, and after separation and purification, active TPI with more than 95% of purity can be obtained. The TPI obtained by the genetic engineering and the TPI naturally separated have the distinction that the high-purity genetic engineering enzyme does not contaminate other relevant enzymes which are symbiotic in plants, and the gene engineering method is suitable for the requirement of specific biological medicine research to pure products.

Description

Genetically engineered prepares the method for spinach chloroplast triose phosphate isomerase

The present invention relates to molecular bioengineering, relate in particular to genetically engineereds such as the regulation and control zone of adopting computer aided design (CAD) genetic expression, gene clone, genetic expression, set up with intestinal bacteria and prepare the very high protein of catalytic efficiencies all in high light efficiency plant-spinach-spinach chloroplast triose phosphate isomerase TPI.

Trisaccharide phosphate isomerase TPI is a kind of enzyme that extensively exists in the organism, it as shown in fig. 1 catalysis glyceraldehyde 3-phosphate G-3-P and the conversion between the di(2-ethylhexyl)phosphate pyruvic alcohol DHAP.TPI has vital role in the metabolism of biology, nearly all pathways metabolism of three sugar phosphoric esters that comprises is all comprising this conversion.As sugared ferment (Glycolysis), glyconeogenesis (Gluconeogenesis), the biosynthesizing of pentose-phosphate pathway (Pentosephosphate pathway) and lipid acid etc., simultaneously, this process still is fixation of C O in the photosynthesis ₂Calvin circulation institute indispensable.TPI exists with the form of two kinds of isozyme in the higher plant.Be present in cytoplasmic TPI, promptly cTPI mainly plays a role in glycolysis-, glyconeogenesis, and is present in the TPI of chloroplast(id), and promptly cpTPI mainly participates in the Calvin circulation.

Document Henze K. in 1994, Schnarrenberger C., Kellermann J. ， ﹠amp; W.1994, the coding gene sequence that Martin has reported spinach chloroplast TPI among the Plant Mol.Biol.26:1961-1973 as shown in Figure 2.From known this prlmary structure of protein, the TPI in various sources is quite conservative, and their tertiary structure and the mode of action also are very close.Its subunit peptide chain has typically (α/β) ₈Building mode, its internal layer are that the parallel βZhe Die of stereotyped writing forms the kernel drum, and the stereotyped writing α spiral that skin is attached thereto tightly twines and forms concentric barrel-like structure, and several big Loop encircle, coiled coil is connected α spiral and βZhe Die with βZhuan Jiao at random.What constitute drum mainly is hydrophobic amino acid, and hydrophobic side chain is positioned at the inboard hydrophobic inner core that forms enzyme of drum, and polare Aminosaeren is positioned at the outside and is exposed among the solvent, and it is inboard that avtive spot is positioned at bucket, is a bag shape.When substrate with after the avtive spot of bag shape combine, one section adjacent peptide ring just covers up substrate, avoiding the attack of water, assurance is only carried out isomerization reaction and hydrolysis reaction is not taken place; At this moment the relevant residue on another subunit also helps water is excluded from the active region, forms hydrogen bond to guarantee substrate and avtive spot.That TPI had was this (α/β) ₈Structure is extensively deposited at nature.That the enzyme of having found to comprise 17 kinds of difference in functionalitys such as the big subunit of Rubisco, zymohexase so far all has is this (α/β) ₈Basic structure, although uncorrelated fully on the function, avtive spot all is present near the C end, is usually located on a βZhe Die and the ring that the α spiral links to each other.

The catalytic efficiency of TPI is very high.The speed of reaction of its isoversion is about 7 alkali fast 10 than a simple pKa ¹⁰About.The rate-limiting step of catalyzed reaction is the velocity of diffusion of substrate and product discrepancy active region.Therefore, TPI has been one and has evolved to such an extent that level off to perfect enzyme in a sense, has not had what selective pressure further to impel it to improve catalytic efficiency.

Based on TPI catalytic activity and typical (α/β) efficiently ₈Tertiary structure, therefore the structure activity relationship as the research enzyme is a good model, its rite-directed mutagenesis combines with crystalline structure and obtains people and pay close attention to widely.And the genetically engineered of higher plant TPI is studied also seldom.Only cloned several genes, reported the corn clone gene among the Marchionni M.and Gilbert W.Cell.46:133-141 as 1986; Nineteen ninety Sato F., Fitchen J.H., Takeshita N., Hashimoto T., Okada N. has reported clone coptis gene among the and Yamada Y.Agric.Biol.Chem.54:2189-2191, and coptis cTPI gene has been carried out simple expression in intestinal bacteria; Paran I. in 1992, Kesseli R.V., Westphal L.and Michelmore R.W.Genome.35:627-635 has cloned the lettuce gene; 1993, reported clone's paddy gene among Xu Y.and Hall T.C.Plantphysiol.106:459-467 in 1994 and the Plant physiol.102:697, and the expression of fusion gene in transgene tobacco of paddy rice cTPI and GRD beta-glucuronidase GUS inquired into; Zhang X.H.and ChinnappaC.C.Genome.17:148-156 had reported clone China pink gene in 1994; Nineteen ninety-five SchmidtM., Svendsen I. ， ﹠amp; Reported clone's oat gene among the Feierabend J.Biochimica et Biophysica Acta.1261:257-264, Henze K. in 1994, Schnarrenberger C., Kellermann J. ， ﹠amp; Reported clone spinach gene among the Martin W.Plant Mol.Biol.26:1961-1973; Reported among the Shih M.PlantPhysiol.194:1103-1104 in 1994 that the clone intends southern shepherd's purse gene; Nineteen ninety-five Ben-Nissan G. ， ﹠amp; Weiss D.J.Plant Physiol.147:58-62 has reported the grand gene of leading a cow of shell; Research that can be present mainly concentrates on the research aspect of tenuigenin TPI, and expresses lower; The chloroplast(id) TPI that obtains higher plant by genetically engineered yet there are no report.Based on chloroplast(id) TPI at Calvin round-robin CO ₂The vital role in fixing and the consideration of organic synthesis aspect, we obtain the TPI gene by PCR method from higher plant, the machine aided design is suitable for the translation initiation region sequence of colibacillus high expression as calculated, change improved gene over to expression system and express, obtain activated TPI albumen by gene engineering method.

Purpose of the present invention is exactly that gene engineering research prepares spinach chloroplast triose phosphate isomerase, can be directly in intestinal bacteria high expression level go out with natural spinach chloroplast triose phosphate isomerase to have same acid sequence and active polypeptide.Promptly from its total mRNA of the leaf of spinach separation and Extraction, with design synthetic oligonucleotide is primer, obtain spinach chloroplast triose phosphate isomerase TPI gene by inverse transcription polymerase chain reaction RT-PCR, correlated expression regulation and control zone by computer aided design (CAD), make between ribosome bind site and the initial code and be rich in AT, make up corresponding cloned plasmids pC1TPIa and expression plasmid pE1TPIa, and change improved expression plasmid over to host e. coli C600 (pcI857) expression system and express, make the expression amount of target protein account for 36% of coli somatic total protein; At last expression product is carried out separation and purification, thereby obtain activated spinach TPI albumen.

The present invention be a kind of can be in intestinal bacteria the gene engineering preparation method of high expression level spinach chloroplast triose phosphate isomerase, it is to obtain highly purified spinach TPI albumen by the separation and purification of the structure of the acquisition of spinach chloroplast triose phosphate isomerase gene, cloned plasmids and expression plasmid, genetic expression, expression product and determination of activity.Concrete preparation method is:

1, the structure of the acquisition of trisaccharide phosphate isomerase TPI structure gene, cloned plasmids and expression plasmid:

With the leaf of spinach is raw material, according to document Henze K., and Schnarrenberger C., Kellermann J. ， ﹠amp; Synthetic PCR primer T-1 and the T-2 that contains the required restriction enzyme recognition sequence of clone accordingly of dna sequence dna design of the Fig. 2 that reports among Martin W. (1994) the Plant Mol.Biol.26:1961-1973, extract total RNA with the Guanidinium hydrochloride method, oligomerization [dT]-cellulose purification mRNA, cDNA first chain, pcr amplification are then synthesized in reverse transcription.Pcr amplification product is through the XbaI/BamHI double enzymolysis, and the LMP agarose gel electrophoresis separates linear fragment, and by the vector plasmid pUC1802 zone accordingly that is cloned into shown in Figure 3, transformed into escherichia coli JM83 obtains containing the cloned plasmids pC0cpTPI of TPI encoding gene.Further identify through sequence analysis, after gene recombination obtains to contain the right-on clone of encoding gene order.Adopt the corresponding expression regulation order of computer aided design (CAD), synthetic has positive and negative burst of fragment of SD order through 5 '-phosphorylation, pairing, BamHI/XbaI double digestion structure gene fragment with plasmid pC0cpTPI, be recombined into vector plasmid pUC1802 zone accordingly, transformed into escherichia coli JM83 obtains containing SD cloned plasmids pC1cpTPI in proper order; This plasmid separates the fragment that contains structure gene through the EcoRI/BamHI double digestion, is recombined into the EcoRI/BamHI cloning site of pPLc2833, and transformed into escherichia coli C600 (pcI857) obtains corresponding expression plasmid pE1TPI as Fig. 3.

Then, making 9 bases by the based composition between PCR method change SD order and the initial code ATG all is A, T; Simultaneously, under guaranteeing the constant prerequisite of amino acid, change the trigram of codon in structure gene 5 '-end order, G, C changes the A of colibacillus high expression codon into as far as possible, T, the design of this PCR primer T-7 and SD order is to be formed with newly-generated the mRNA 5 '-end that guarantees hypothesis that to be beneficial to rrna be prerequisite in conjunction with the secondary structure of SD order.At first, plasmid pC1cpTPI makes linear fragment through the EcoRI/BamHI double enzymolysis, is template with this fragment, and T-7 and T-2 are that primer carries out pcr amplification.The PCR product has the fragment T-5 of SD order with synthetic behind the BamHI enzymolysis, T-6, carrier linear plasmid EcoRI/BamHI flush end connect back transformed into escherichia coli JM83, obtain pC1TPIa.The chloroplast(id) TPI structure gene that will have the SD order is recombined into the expression plasmid pE1TPIa that the pPLc2833 corresponding position obtains chloroplast(id) TPI.

Cloned plasmids pC1TPIa host cell intestinal bacteria JM83:1999 China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on June 18, preserving number is CGMCC NO.0401.

Expression plasmid pE1TPIa host cell intestinal bacteria C600 (pcI857): China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on June 18th, 1999, preserving number is CGMCC NO.0400.

2, the expression of spinach chloroplast TPI:

Expression plasmid pE1TPIa gets behind the transformed into escherichia coli C600 (pcI857) to dilute after 30 ℃ of joltings of single bacterium colony access LB substratum are spent the night and cultivates, and thermal induction was expressed 4 hours, and as shown in Figure 4, expression product can reach about 36% of total protein concentration.

3, the separation and purification of expression product and determination of activity:

Adopt purification step as shown in Figure 7, the ultrasonication cell, the Vetstrep precipitate nucleic acids, the ammonium sulfate precipitation preliminary purification, through the separation of DEAE-Sepharose Fast Flow ion exchange column target protein purity is reached more than 90%, further through Sephadex G-75 gel-filtration such as Fig. 9, purity is greater than 95%, as shown in figure 10.

Description of drawings:

The catalytic reaction of Fig. 1: TPI;

Fig. 2: spinach chloroplast triose phosphate isomerase gene and amino acids coding thereof

The RT-PCR primer of order and design;

Fig. 3: the construction procedures figure of acquisition of TPI structure gene and cloned plasmids and expression plasmid;

Fig. 4: the thermal induction expression of results figure of expression plasmid pE1TPIa;

Illustrate among the figure: (pE1TPIa, nutrient solution pcI857) sway 3 hours to A at 30 ℃ to contain C600 _650nmReach 0.7, elevated temperature to 42 ℃ abduction delivering.Different induction time samplings, sample is after 15% SDS-PAGE separates, and albumen dyes with Coomassie brilliant blue G-250.Induction time is respectively: swimming lane 3:0min; Swimming lane 4:15min; Swimming lane 5:30min; Swimming lane 6:60min; Swimming lane 7:120min; Swimming lane 8:240min.(pcI857 pPLc2833) induced under 42 ℃ 4 hours swimming lane 1:C600.Swimming lane 2: molecular weight standard, Trichosanthin, 27.2kD.

The two groups of expression plasmid pE1TPIa of Fig. 5: TPI and pE1TPI are in SD order and initial

The difference of nucleotide sequence between the codon;

The two groups of expression plasmid pE1TPIa of Fig. 6: TPI and the abduction delivering result of pE1TPI

Relatively;

Illustrate among the figure: contain the nutrient solution of the bacterium C600 (pcI857) of expression plasmid, the same terms was expressed 4 hours down, got same amount and carried out electrophoresis.Sample after 15% SDS-PAGE separates, swimming lane 1:C600 (pcI857, pE1TPI) 30 ℃ of overnight incubation are not induced; Swimming lane 2:C600 (pcI857, pPLc2833) 30 ℃ were swayed 3 hours, induced 4 hours for 42 ℃; Swimming lane 3: molecular weight standard, 27.2kD; Swimming lane 4,5:C600 (pcI857, pE1TPI) abduction delivering; Swimming lane 6,7:C600 (pcI857, pE1TPIa) abduction delivering.

Fig. 7: the TPI separation and purification schema of expression;

Fig. 8: the figure as a result of DEAE-Sepharose Fast Flow column chromatography purification TPI;

Illustrate among the figure: DEAE-Sepharose fast flow post is 3.5 * 15cm, buffer A: 10mmol/l K ₂HPO ₄-KH ₂PO ₄, pH=7.6,1mmol/l EDTA, 20mmol/l beta-mercaptoethanol, 0.02% NaN ₃, be the KCl linear gradient leaching of the 0.1-0.3mol/l of base fluid with the 600ml buffer A." ● ": A _280nm" ■ ": enzyme activity U/ml.

Fig. 9: the figure as a result of Sephadex G-75 column chromatography purification TPI;

Illustrate among the figure: Sephadex G-75 post is 2.1 * 100cm, elution buffer: 10mmol/l K ₂HPO ₄-KH ₂PO ₄, pH=7.6,1mmol/l EDTA, 20mmol/l beta-mercaptoethanol, 0.02% NaN ₃, " ● ": A _280nm, " zero ": enzyme activity U/ml.

Figure 10: the SDS-PAGE analysis chart in the TPI purge process;

Illustrate among the figure: sample separates through 15% SDS-PAGE, and swimming lane 1:C600 (pPLc2833, pcI857); Swimming lane 2: whole bacterial protein; Swimming lane 3: cell pyrolysis liquid; Swimming lane 4: Vetstrep post precipitation supernatant; Swimming lane 5: supernatant behind the ammonium sulfate precipitation; Behind the swimming lane 6:DEAE-Sepharose Fast Flow column chromatography purification; Behind the swimming lane 7:Sephadex G-75 column chromatography purification.

Lineweaver-Burk double-reciprocal plot method is measured TPI under Figure 11, the 25 ℃ of conditions

The Km value

The present invention compared with the prior art, the at present TPI genetic engineering research of bibliographical information seldom, and expression is low. Can express efficiently the TPI of plant origin by Escherichia coli fermentation, but high productivity obtains not stain the highly purified active TPI of other relevant enzyme of symbiosis in the plant. By CAD correlated expression regulation and control zone, be rich in the replacement in AT zone so that expression reaches 36% between ribosome bind site and the initial numeral. Can prepare highly purified TPI by common biofermentation. Following examples help to understand the present invention, but are not limited to content of the present invention

Materials and methods:

Fresh spinach is available from the food market. Get fuller vanes liquid nitrogen flash freezer after cleaning and in-70 ℃ of refrigerators, preserve.

Lysozyme, p-Methyl benzenesulfonyl fluorine, Dowex-1 type anion exchange resin, Dowex 50H⁺Type ion exchange resin, acrylamide, glyceraldehyde 3-phosphate dehydro-genase GAPDH, α-GDH and ester of Harden Young FBP are all available from Sigma company;

PUC18 is available from Pharmacia Biotech company

T ₄Dna ligase, NADH, NAD⁺, bag filter, streptomycin sulphate, efficient oligomerization (dT)-cellulose and placenta RNA enzyme inhibitor are available from Huamei Bio-Engrg Co.;

The Taq archaeal dna polymerase, lauryl sodium sulfate SDS, the AMV reverse transcriptase is available from Boehringer-Mannheim company;

Sep-pak C18 reversed-phase column is available from Waters company;

Low melting-point agarose gel (LMP Agarose) is available from GIBCO BRL;

SeaPlaque GTG Agarose is available from FMC Corp.;

Protein quantification reagent is available from Bio-Rad company;

Sephadex G-75, DEAE-Sepharose fast flow resin is available from PharmaciaBiotech company;

Beta-mercaptoethanol is available from Fluka company;

Sodium ampicillin Amp and sulphuric acid kanamycin Kana are available from Shanghai the 4th Pharmacy stock Co., Ltd;

PEG-6000 is available from Shanghai reagent one factory;

Ultra-filtration membrane XPH-5 is available from Shanghai Wu Jing separate apparatus factory.

Join solution all with heavily steaming distilled water, all experimental wares all pass through high pressure steam pot sterilization;

Genetically engineered fructose-1,6-diphosphate zymohexase by this laboratory according to the old Hypon of document, Yang Chunsong, Acta Biochimica et Biophysica Sinica, 1997,29:613-616 preparation.

Synthetic, the detection and quantitative of substrate glyceraldehyde 3-phosphate G-3-P:

Synthetic: the 20mmol/l of 150ml, contain 1gFBP8H in the EDTA reaction system of pH7.4 ₂O and 4ml heavily steam acetaldehyde, and 180～200 units are purifying and do not contain the fructose 1 of TPI, the 6-bisphosphate aldolase, and 37 ℃ of reactions 20 minutes down, the ice bath cooling, directly through 1 * 10cm, HCOO ^-The Dowex-1 anion-exchange column of type is with 1 liter of H ₂O-HCOONH ₄(0.25mol/l)/HCOOH (0.1mol/l) gradient elution.With GAPDH and NAD ⁺Detect, collect the product component, add the CaCl of 2 mol ratios ₂The dehydrated alcohol of solution and 2 times of volumes, 0 ℃ precipitates 2 hours.Centrifugal collecting precipitation, 80% cold washing with alcohol, the anhydrous diethyl ether washing, drying under reduced pressure, quantitatively ,-70 ℃ of preservations.

Detect and reach quantitatively: contain 100mmol/l in the 1ml reaction system, the Tris-HCl of pH7.5, the EDTA of 1mmol/l, the NAD of 1mmol/l ⁺, the Na of 5mmol/l ₂AsO ₄, the GAPDH of 0.15mg/ml adds behind the G-3-P to be measured and surveys A in 25 ℃ _340nm

Substrate di(2-ethylhexyl)phosphate pyruvic alcohol DHAP presses document Effenberger, F.and Straub, A., Tetrahedron Lett.1987, the method preparation of 28:1641-1644.

DHAP's is quantitative:

Reduced liquid: 0.2g 1-amino-2-naphthol-4-sulfonic acid, 1.2g NaHSO ₃And 1.2gNa ₂SO ₃Mix the back and fully grind, get this mixture of 0.25g before the use and be dissolved in the 10ml water stand-by.

The KH of reference liquid: 10mmol/l ₂PO ₄The aqueous solution.

The drafting of typical curve: in vitro add reference liquid 0.1-1ml at 10 respectively and respectively add the H that 1ml concentration is 2.5mol/l ₂SO ₄With 3 concentration HNO that is 2mol/l ₃And 1ml concentration is 2.5% ammonium molybdate, and dilution a little adds the 0.1ml reduced liquid behind the mixing, is diluted to 10ml, places and surveys A after 10 minutes _660nm

The mensuration of sample: get different each 1ml of dilution sample, adding 1ml concentration is the H of 2.5mol/l ₂SO ₄, on flame, being heated to the little Huang of liquid, the cooling back adds 3 HNO that concentration is 2mol/l ₃, continue to be heated to foam and occur, cool off the back and add 1ml water, boiling water bath insulation 5 minutes, cooling, the ammonium molybdate of adding 1ml 2.5% adds the 0.1ml reduced liquid behind the mixing, be diluted to 10ml, places and surveys A after 10 minutes _660nmConcentration according to the typical curve calculation sample.

* Luria-Bertani substratum: contain peptone (Tryptone) 10g in every liter, yeast extract is 5g, and sodium-chlor 10g adds NaOH solution adjust pH to 7.2, constant volume.Add 15g (1.5% w/v) agar when joining solid LB substratum again, autoclaving.

Other inorganic salt and chemical reagent commonly used are commercially available homemade analytical reagent.

Testing required oligonucleotide fragment is synthesized and purifying on dna synthesizer by the phosphoramidite triester method by this laboratory;

T-1：AC CTC TAG ATG GCT GGC TCT GGA AAG TT

T-2：AC GGG ATC CTA TCA AGC AGC AAC TTT CTT TGC

T-3：AAT TCG GTA AGG AGG TGA CCT

T-4：CTA GAG GTC ACC TCC TTA CCG

T-5：AAT TCA GTA AGG AGG TTA TTT

T-6：AAA TAA CCT CCT TAC TG

T-7：A AAT ATG GCT GGC TCT GGT AAG TTC TTC GTT GGT

Bacterial classification and plasmid:

Vector plasmid pPLc2833 and intestinal bacteria C600 (pcI857) are that Massachusetts Institute of Technology professor Khorana provides; Vector plasmid pE1Rfbp is made up according to document " Chen Haibao, Yang Chunsong, Acta Biochimica et Biophysica Sinica, 1997,29:613-616 " by this laboratory; Plasmid pWR13 and intestinal bacteria JM83 see document " Chen, H.-B., etal., Nucl.Acids Res, 1990,18:871-878 " introduction; PUC1802 and pUC1801 are the plasmid of deriving after pUC18 changes the polylinker district.

Embodiment 1

The structure of acquisition, cloned plasmids and the expression plasmid of trisaccharide phosphate isomerase TPI structure gene:

The a.RNA extracting, mRNA purifying, reverse transcription

From-70 ℃ of refrigerators, take out 10g the leaf of spinach, adopt Guanidinium hydrochloride method extracted total RNA, further get total mRNA through (dT) n-cellulose purification.

With 30 μ l reverse transcription damping fluids, damping fluid is by 50mmol/l in the ice bath, the TrisHCl of pH8.3, the MgCl of 8mmol/l ₂, 30mmol/l the DTT of KCl, 1mmol/l form, four kinds of 2 '-deoxynucleoside-5 '-triphosphoric acids of 1mmol/l, 250ng Oligo (dT), the AMV ThermoScript II of 20 unit placenta RNA enzyme inhibitorss and 6mg mRNA and 25 units was reacted 1 hour down at 42 ℃.Reverse transcription product made product sex change and deactivation ThermoScript II in 5 minutes in 95 ℃ of water-baths, was diluted with water to 100 μ l then in-20 ℃ of preservations.

With 6 μ l 5x reverse transcription damping fluids, damping fluid is by 250mmol/l, pH8.3TrisHCl, 40mmol/l MgCl ₂, 150mmol/l KCl, 5mmol/l DTT form, 6 μ l respectively are four kinds of 2 '-deoxynucleosides-5 '-triphosphoric acid mixed solution of 5mmol/l, the 250pg/ μ l oligomerization (dT) of 1 μ l, 20 unit placenta RNA enzyme inhibitorss and 6mg mRNA add respectively in the 0.5ml centrifuge tube, the AMV ThermoScript II that adds 25 units at last, water is mended to 30 μ l, reacts 1 hour down in 42 ℃ behind the mixing gently.Reverse transcription product made product sex change and deactivation ThermoScript II in 5 minutes in 95 ℃ of water-baths, was diluted with water to 100 μ l then in-20 ℃ of preservations.

The pcr amplification of b.cDNA

With 100 μ l pcr amplification damping fluids, damping fluid is by 10mmol/l in the special-purpose reaction tubes of PCR, the TrisHCl of pH8.3,1.5mmol/l MgCl ₂, 50mmol/l KCl form 5 ' of four kinds of 2 '-deoxynucleosides-5 ' of 2mmol/l-triphosphoric acid mixed solution 5pmol-and 3 '-primer; The above-mentioned cDNA of 50ng, 95 ℃ of water-baths are 5 minutes behind the mixing, add 5 Taq of unit archaeal dna polymerases again, are covered on the reaction mixture with 100 μ l light mineral oil, centrifugal a little laggard performing PCR circulation.The PCR condition is: 94 ℃ of sex change 1 minute, and 50 ℃ of pairings 2 minutes, 72 ℃ were extended 1.5 minutes; Totally 30 take turns circulation, end wheel circulation was extended 10 minutes.Get 5 μ l and separate the back ethidium bromide staining with agarose gel electrophoresis, ultraviolet lamp detects down.

C. the structure of cloned plasmids and expression plasmid

The PCR product is chloroform-primary isoamyl alcohol mixed solution extracting of 24: 1 through volume ratio, 2 volume dehydrated alcohols precipitation, 75% cold washing with alcohol, be dissolved in behind the drying under reduced pressure in the suitable damping fluid, through XbaI and BamHI double enzymolysis, the LMP sepharose separates the back by 3: 1 ratio and adding 0.3 T4DNA of unit ligase enzyme after carrier linear plasmid pUC1802 through XbaI and BamHI double enzymolysis mixes, transformed into escherichia coli JM83 according to a conventional method after 16 ℃ of connections are spent the night obtains containing the cloned plasmids pC0cpTPI of TPI encoding gene.Further identify through sequence analysis, after gene recombination obtains to contain the right-on clone of encoding gene order.Adopt the corresponding expression regulation order of computer aided design (CAD), synthetic has positive and negative burst of fragment of SD order through 5 '-phosphorylation, pairing, BamHI/XbaI double digestion encoding gene fragment with plasmid pC0cpTPI, be recombined into vector plasmid pUC1802 zone accordingly, transformed into escherichia coli JM83 obtains containing SD cloned plasmids pC1cpTPI in proper order; This plasmid separates the fragment that contains structure gene through the EcoRI/BamHI double digestion, is recombined into the EcoRI/BamHI cloning site of pPLc2833, and transformed into escherichia coli C600 (pcI857) obtains corresponding expression plasmid pE1TPI as Fig. 3.

Then, making 9 bases by the based composition between PCR method change SD order and the initial code ATG all is A, T; Simultaneously, under guaranteeing the constant prerequisite of amino acid, change the trigram of codon in structure gene 5 '-end order, G, C changes the A of colibacillus high expression codon into as far as possible, T, the design of this PCR primer T-7 and SD order is to be formed with newly-generated the mRNA5 '-end that guarantees hypothesis that to be beneficial to rrna be prerequisite in conjunction with the secondary structure of SD order.At first, plasmid pC1cpTPI makes linear fragment through the EcoRI/BamHI double enzymolysis, is template with this fragment, and T-7 and T-2 are that primer carries out pcr amplification.The PCR product has the fragment T-5 of SD order with synthetic behind the BamHI enzymolysis, T-6, carrier linear plasmid EcoRI/BamHI flush end connect back transformed into escherichia coli JM83, obtain pC1TPIa.The chloroplast(id) TPI structure gene that will have the SD order is recombined into the expression plasmid pE1TPIa that the pPLc2833 corresponding position obtains chloroplast(id) TPI.

Embodiment 2

Genetic expression:

Expression plasmid pE1TPIa, transformed into escherichia coli C600 (pcI857).Get single bacterium colony and insert small volume LB substratum, contain Amp 100 μ g/ml in this substratum, Kana 50 μ g/ml, jolting is spent the night under 30 ℃ of conditions.Adopt 3 liters of shake-flask culture then under without situation about express optimizing, go up 1 and state in the nutrient solution by inoculation in 1: 100, be cultured to the logarithmic growth middle and later periods under 30 ℃ of conditions, through 42 ℃ abduction deliverings 4～6 hours, expression product detected with 15%SDS-PAGE.

Embodiment 3

The expression product separation and purification:

Under 4 ℃ of conditions, the coli somatic of centrifugal collection behind abduction delivering, be resuspended in the 20ml lysis buffer, this damping fluid is by 100mmol/l, the Tris-HCl of pH7.5, the EDTA of 2.5mmol/l, the beta-mercaptoethanol of 20mmol/l, the 10mg N,O-Diacetylmuramidase, the PMSF of 0.5mmol/l forms, and the ultrasonic disruption cell is 5 minutes in ice bath.15, centrifugal 30 minutes of 000g collects lysate.Adding Vetstrep to final concentration under the condition of ice bath is 1%, stirs 30 minutes postprecipitation nucleic acid.15, centrifugal 15 minutes of 000g, get supernatant, slowly add ammonium sulfate to 45% saturation ratio down in ice bath, agitation condition, continue to stir 15～30 minutes, left standstill 15～30 minutes, 15, centrifugal 15 minutes of 000g, adding ammonium sulfate to saturation ratio in supernatant liquor again is 85%, handles to such an extent that precipitate with above-mentioned condition.Then with this precipitation with the dissolving of the damping fluid of proper volume and to dialysed overnight, this damping fluid is by 10mmol/l, the K of pH7.6 ₂HPO ₄-KH ₂PO ₄, the EDTA of 1mmol/l, the beta-mercaptoethanol of 20mmol/l is formed.

Dialyzate is centrifugal, gets the supernatant liquor application of sample to the DEAE-Sepharose Fast Flow weak anion exchange resin of crossing through the buffer A balance, and wherein buffer A liquid is 10mmol/l, the K of pH7.6 ₂HPO ₄-KH ₂PO ₄, the EDTA of 1mmol/l, the beta-mercaptoethanol of 20mmol/l and 0.02% NaN ₃Form.Use buffer A drip washing to A earlier _280nm＜0.03, be the KCl linear gradient leaching of the 0.1-0.3mol/l of base fluid with the 600ml buffer A then, press the speed of 4.0ml/ pipe/4min and collect product, the ultraviolet detection elution peak, the result as shown in Figure 8, SDS-PAGE detects purity of protein.

The above-mentioned target protein elutriant that contains is merged, and to about 3ml, last sample is to Sephadex G-75 post with ultra-filtration membrane XPH-5 ultrafiltration and concentration, with the buffer A wash-out, and the ultraviolet detection elutriant, SDS-PAGE detects purity of protein, the results are shown in accompanying drawing 9.

Embodiment 4

Protein-active is measured:

With glyceraldehyde 3-phosphate G-3-P is that substrate: 1ml survey reaction system alive contains 100mmol/l, the Tris-HCl of pH7.5, the EDTA of 5mmol/l, 0.2mmol/l NADH, 19 μ g alpha-phosphate glycerol dehydrogenases, the G-3-P of 0.5mmol/l, and survey A in 25 ℃ after adding the TPI of 1-2ng to be measured _340nmThe variation of place's absorbancy.

With di(2-ethylhexyl)phosphate pyruvic alcohol DHAP is to contain 100mmol/l, the Tris-HCl of pH7.5, the EDTA of 1mmol/l, the NAD of 1mmol/l in substrate: the 1ml survey live body system ⁺, the Na of 5mmol/l ₃AsO ₄, 0.15mg glyceraldehyde 3-phosphate dehydro-genase GAPDH, the DHAP of 1.2mmol/l surveys A in 25 ℃ after adding 50-100ng TPI to be measured _340nmThe variation of place's absorbancy.

Adopt the activity tracking and the purifying yield of above-mentioned separation purification method to be listed in the table below.

Purification step	Total protein concentration (mg)	Total activity (u) G-3-P DHAP		Than (u/mg) G-3-P DHAP that lives		Yield (%) G-3-P DHAP
								Broken bacterium supernatant	278	385851	9839	1388	35.4	100	100
The Vetstrep precipitation	253	354846	9194	1403	36.3	92.0	93.4
								Ammonium sulfate precipitation	138	324749	8650	2353	62.7	84.2	87.9
DEAE-Sepharose F.F.	67.3	301145	7723	4475	115	78.0	78.5
								Sephadex G-75	57.6	285643	7313	4959	127	74.0	74.3

Annotate: wherein the enzyme of separation and purification comes from 1 liter of LB substratum, and cell is through ultrasonication, and all purification steps all carry out in 4 ℃.Unit of enzyme is defined as: under the saturated situation of substrate, 25 ℃ of per minutes consume glyceraldehyde 3-phosphate or the di(2-ethylhexyl)phosphate pyruvic alcohol of 1 μ mol.

Embodiment 5

The mensuration of the enzymatic analysis-Michaelis-Menton constant of expression product:

Adopting Lineweaver-Burk double-reciprocal plot method, is substrate with glyceraldehyde 3-phosphate G-3-P and di(2-ethylhexyl)phosphate pyruvic alcohol DHAP respectively, at 25 ℃ of zymetology constants that record the spinach chloroplast TPI of this genetically engineered gained, as Figure 11.

When being substrate with G-3-P, Km _(G-3-P)Value is 0.68mmol/l, Vmax _(G-3-P)Be 3.16 * 10 ⁴μ mol/minmg, Kcat _(G-3-P)Be 4.51 * 10 ³/ S; When being substrate with DHAP, Km _(DHAP)Value is 7.27mmol/l, Vmax _(DHAP)Be 1.04 * 10 ³μ mol/minmg, Kcat _(DHAP)Be 1.16 * 10 ²/ S.

Claims (6)

1, a kind of genetically engineered prepares the method for spinach chloroplast triose phosphate isomerase, after it is characterized in that obtaining natural TPI gene, by computer aided design (CAD) this gene is transformed and to be cloned matter position and expression plasmid accordingly, go out spinach chloroplast triose phosphate isomerase at expression in escherichia coli then.

2, genetically engineered as claimed in claim 1 prepares the method for spinach chloroplast triose phosphate isomerase, it is characterized in that concrete preparation process is

A. the acquisition of spinach chloroplast triose phosphate isomerase gene;

B. the structure of cloned plasmids and expression plasmid;

C. genetic expression;

D. the separation and purification of expression product.

3, genetically engineered as claimed in claim 1 or 2 prepares the method for spinach chloroplast triose phosphate isomerase, it is characterized in that structure gene is partly with the expression regulation zone that helps high expression level of computer aided design (CAD) among constructed cloned plasmids pC1TPIa and the expression plasmid PE1TPIa.

4, genetically engineered as claimed in claim 3 prepares the method for spinach chloroplast triose phosphate isomerase, it is characterized in that described expression vector is pPLc2833.

5, genetically engineered as claimed in claim 3 prepares the method for spinach chloroplast triose phosphate isomerase, it is characterized in that described expression host cell is intestinal bacteria C600 (pcI857).

6, genetically engineered as claimed in claim 1 or 2 prepares the method for spinach chloroplast triose phosphate isomerase, it is characterized in that in the genetic expression process, the expression amount of target protein accounts for 36% of coli somatic total protein.

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