CN103436511B - High temperature alkaline protease and preparation method thereof - Google Patents
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Abstract
The invention provides a high temperature alkaline protease and a preparation method thereof, and relates to a protease, particularly to a high temperature alkaline protease Fppro1 and a preparation method thereof, and high temperature alkaline protease gene Fppro1. The production strain of the high temperature alkaline protease Fppro1 is Flammeovirga Pacifica H2. During preparation, the high temperature alkaline protease gene Fppro1 is cloned, and is inserted into an expression vector to construct a recombinant expression vector carrying the high temperature alkaline protease gene Fppro1; the recombinant expression vector carrying the high temperature alkaline protease gene Fppro1 is transformed into escherichia coli BL21, and positive clones are selected and cultured in a culture medium; and the fermented escherichia coli BL21 cells are collected through centrifugation, the escherichia coli BL21 cells are resuspended in a lysis buffer, the lysed suspension is centrifuged, the supernatant is collected and is mixed with Ni-NTA Agarose, and purification is performed.
Description
Technical field
The present invention relates to proteolytic enzyme, particularly a kind of high temprature detectation and preparation method thereof.
Background technology
Sumizyme MP refers to the enzyme of protein hydrolysate peptide bond within the scope of pH value meta-alkalescence, many from bacillus protein enzyme at present, major part belongs to serine protease, and it can generate polypeptide or amino acid by hydrolyze protein molecules peptide chain, has the ability of stronger decomposing protein.
Sumizyme MP is found in Pancreas Sus domestica the earliest, 1913, and first islet proteins enzyme is used for washing soaking agent by Rohm.Jaag in 1945 etc. have found Sumizyme MP in microorganism, and the research and apply of this enzyme is rapidly developed.1963, Novo Nordisk Co., Ltd (existing Novozymes Company) found efficiently, is applicable to the Sumizyme MP Alcalase of washing composition.Sumizyme MP commodity at present for washing composition also get more and more, Sabinase, Kannase, Durazyme etc. of such as Novozymes Company, the Properase CT of the KAP of KAO. Corp. SA, GENENCOR company, the Savinase4.OT high temprature detectation etc. of NOVO company.At present, proteolytic enzyme is class of enzymes that should be with the most use in industrial enzymes, and account for 60% of enzyme total amount, wherein Sumizyme MP just account for 25%.Except washing composition, the industry such as it is also widely used in food, medical treatment, brewages, silk, process hides.Its application prospect in business, and the vital role in scientific research makes it obtain the long-term concern of researchist and domestic and international company.
Optimum temperature warm Sumizyme MP and optimum temperature in 30 ~ 40 DEG C is the most common in the patent of current research report and application/authorize lower than the low-temperature alkaline protease of 30 DEG C.And the research of the high temprature detectation of optimum temperature more than 50 DEG C is reported and the patent of application/mandate is less.Chinese patent CN103013960A provides a kind of high temprature detectation and recombinant expressed engineering bacteria thereof, and its optimum temperature is 60 DEG C, and optimum pH is 12.Enzyme, with the addition of after various stablizer makes mixed preparation, is deposited the residual enzyme vigor still having 95% for 30 days, but is not provided the thermal stability characteristics of protoenzyme at 40 DEG C.Chinese patent ZL200620111116.8 provides a kind of refractory metal prolease gene engineering bacterium and preparation method thereof, and its optimum temperature is 65 DEG C, and optimum pH is 7.2, does not belong to Sumizyme MP, does not provide the feature of thermostability.Chinese patent ZL200610069339.2 provides a kind of bacillus thuringiensis screening and cultural method of high-yield thermostable proteinase, wherein said proteolytic enzyme optimum temperature is 55 DEG C, the basic non-inactivation of 1h under 55 DEG C of conditions, under 60 DEG C of conditions, 1h still has the vigor of more than 60%, does not provide the feature of action pH value.
High temprature detectation at high temperature can play maximum catalytic efficiency, has good thermostability, has using value widely than middle gentle low-temperature alkaline protease.In actual applications, except the vigor of enzyme under differing temps, condition of different pH, the stability of enzyme is one of key index of evaluate application potentiality, comprises thermostability, tolerance etc. to organic solvent.Therefore, alkalescence, thermostable high-temperature proteolytic enzyme have important development and application values.
Summary of the invention
The present invention aims to provide a kind of high temprature detectation gene Fppro1.
Second object of the present invention is to provide a kind of recombinant high temperature Sumizyme MP Fppro1 encoded by described high temprature detectation gene Fppro1.
3rd object of the present invention is to provide the preparation method of a kind of described recombinant high temperature Sumizyme MP Fppro1.
Described high temprature detectation called after Fppro1, it produces bacterial strain is Pacific Ocean heat color bacillus (FlammeovirgaPacifica) H2, this bacterial strain is preserved in China typical culture collection center on June 13rd, 2012, address: China. Wuhan. Wuhan University, deposit number is: CCTCC NO:M2012229.
The sequence of described high temprature detectation gene Fppro1 is as follows:
1 ATGAAAAATA CAATTTCAAA AATCTTATTG GGTGCTGCTA TTTTTACCCA TATAGGTTTT
61 ACTGCAAACG CACAAGAAGA ACCTCGTAAA GAAGCTCCAA AGAATTGGTT TAACCTTAGC
121 TATGAACAAG ATGGTGTTTA TGGAGTTGGA ACCGAAAGAG CATACGACGA GATTTTAAAA
181 AATAAAAAAT CAAAAAAAGT TGTGGTTGCA GTGATTGATA GCGGAATCGA TATCGATCAT
241 GAAGATCTTA AGGACGTTAT CTGGAAAAAT AAAAAGGAAG TTGCAGGTAA CGGTAAAGAT
301 GATGATAACA ACGGTTATGT TGATGACGTA AACGGTTGGA ACTTTATTGG TGGTGCTGAT
361 GGATCAATGG TGAATGAAGA AAACCTTGAG GTAGCTCGTC TTTATGGAAA ACTTAGTAAG
421 AAGTTTGAAG GAGTAGCTGA AGGTGATGTA GCAAAAGCGG ATAAAAAAGA ATACACTTTG
481 TGGTTAGAAG TGAAAAAAGC TTTCGAAGAA GGCTACACTA AAGCAGAAGA AAACTATACA
541 CGTTACTCTA CATATTTACA CCAATTTCAA AGAGGAAAAG CATTATTTAT GGCTTATTTC
601 GATTTAGAAG ATGAAGGTGA AATTGTTGGT GCGTTAGAAT CATTTGATTC TAGTGATGAA
661 GTACTGATGG GAATGAAGGA GATGACATTA GGTCTTCTTT CACAAGGTGT TACAGATGAT
721 CAGCTTCAAG AAGGTGTTGA CTACTTTGAA GGTCAGGTGA AATCCAATTA TAATTATGAG
781 TTAAATACTC GTGAAATTGT TGGTGATGAT ATCGACAATA AGTCTCAAAG AGATTATGGT
841 AATAATAAAG TAACAGGTCC TGATGCTCTT CATGGTACTC ATGTTGCTGG AATTATTGCT
901 GCCTCAAGAG GAAATGAAAT TGGTATGGAT GGTGTTGCCG ACAATGTAGA AATTATGGTT
961 GTCAGAACTG TACCTAACGG TGATGAACGT GATAAAGATG TTGCAAATTC AATTATTTAT
1021 GCTGTTGATA ATGGTGCACA AATCATTAAT ATGAGCTTTG GTAAACCTTA TTCGCCATAC
1081 AAAGGAACAG TAGATAAAGC TGTTAAGTAT GCTGAATCAA AAGGAGTACT TCTAGTACAT
1141 GCCGCTGGTA ATGACCATAA GAATACAGAT AAAGGAAATA ATTTCCCAAG AGATAAATAT
1201 GATTCTGGCA AAACTGCAAA GAATTGGATA GAGGTTGGAG CATCAACTTG GATGACTGAT
1261 GAGAAACTCC CTGCAACGTT TTCTAACTAT GCTAAGAAAA GTGTAGACTT ATTTGCTCCA
1321 GGTTTTGATA TTTACTCAAC AATTCCAGGT TCAGAATATA CTAATTTAAA TGGTACAAGT
1381 ATGGCATGTC CTGTTGTTGC AGGTTGTGCA GCTGTATTAA TGTCATATTA TCCGAACTTG
1441 TCAGCAGTAC AAGTGAAGAA GATTTTAATG AAAACAGTAA CTCCATTGAA GAGTAAGGAA
1501 GTATTGTTAC CTGGTTATAA TAGTTTAGAA GAAGGTGAAG AGCCTCAAAA AGTTAAATTT
1561 GGATCATTAT CAGTAAGTGG TGGTGTAGTT AACTTATATG AAGCTGTAAA ATACGCTGAA
1621 AAGATCTCAA AATAG
The aminoacid sequence of the high temprature detectation gene Fppro1 that described high temprature detectation gene Fppro1 encodes is as follows:
1 MKNTISKILL GAAIFTHIGF TANAQEEPRK EAPKNWFNLS YEQDGVYGVG
51 TERAYDEILK NKKSKKVVVA VIDSGIDIDH EDLKDVIWKN KKEVAGNGKD
101 DDNNGYVDDV NGWNFIGGAD GSMVNEENLE VARLYGKLSK KFEGVAEGDV
151 AKADKKEYTL WLEVKKAFEE GYTKAEENYT RYSTYLHQFQ RGKALFMAYF
201 DLEDEGEIVG ALESFDSSDE VLMGMKEMTL GLLSQGVTDD QLQEGVDYFE
251 GQVKSNYNYE LNTREIVGDD IDNKSQRDYG NNKVTGPDAL HGTHVAGIIA
301 ASRGNEIGMD GVADNVEIMV VRTVPNGDER DKDVANSIIY AVDNGAQIIN
351 MSFGKPYSPY KGTVDKAVKY AESKGVLLVH AAGNDHKNTD KGNNFPRDKY
401 DSGKTAKNWI EVGASTWMTD EKLPATFSNY AKKSVDLFAP GFDIYSTIPG
451 SEYTNLNGTS MACPVVAGCA AVLMSYYPNL SAVQVKKILM KTVTPLKSKE
501 VLLPGYNSLE EGEEPQKVKF GSLSVSGGVV NLYEAVKYAE KISK
The preparation method of described high temprature detectation gene Fppro1 comprises the following steps:
1) high temprature detectation gene Fppro1 is cloned;
2) high temprature detectation gene Fppro1 is inserted expression vector, build the recombinant expression vector carrying described high temprature detectation gene Fppro1;
3) recombinant expression vector is transformed in intestinal bacteria (E.coli) BL21;
4) choose the positive colony transformed in rear E.coli BL21 and carry out fermentation culture in substratum;
5) the E.coli BL21 cell after collected by centrifugation fermentation, resuspended described E.coli BL21 cell carries out cracking in lysis buffer;
6) by centrifugal for the suspension after cracking in step 5), collect supernatant liquor, then mix with Ni-NTA Agarose, carry out purifying according to purification kit specification sheets, obtain high temprature detectation gene Fppro1.
In step 2) in, described expression vector can be selected from pCold I carrier etc.
In step 4), described substratum is optional from the LB substratum containing 100 μ g/mL penbritins, and the condition of described fermentation culture can be: temperature is 37 ° of C, and shaking table is cultured to A
600when=0.6, then to add isopropylthio-β-D-galactoside (IPTG) to final concentration be 50 μm of ol/L, under 16 ° of C conditions, induce 12h.
In step 5), described lysis buffer formula can be: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/LNaH
2pO
4, pH8.0.
In step 6), described centrifugal condition can be 18000 × g, centrifugal 20min, 4 DEG C.
The present invention has following outstanding advantages:
1. described in, high temprature detectation Fppro1 has new gene order, does not have similar complete ORF gene order in ncbi database.
2. high temprature detectation Fppro1 described in can in E.coli stably express, after induction, the vigor of enzyme can reach 38600U/mL in supernatant liquor.The optimum temperature of recombinase Fppro1 is 60 DEG C, can keep the vigor of more than 60 DEG C, keep the vigor of about more than 90% within the scope of 50 ~ 65 DEG C within the scope of 40 ~ 70 DEG C, has wider operative temperature scope.
3. recombinase Fppro1 has good thermostability: under the condition not adding any stablizer, at 30 DEG C, at least keep 24h non-inactivation; Enzyme activity residue more than 95% after 12h at 40 DEG C; After being incubated 1h at 50 DEG C, vigor has rising slightly on the contrary, and after 2h, vigor keeps about 75% after can keeping 85%, 4h; 60 DEG C are incubated the vigor that 1h still can keep about 65%.
4. Optimun pH is 9, can keep the vigor of more than 80% in the scope of pH8 ~ 10; The vigor of about 65% can be kept when pH7, there is wider pH sphere of action.
Due to above-mentioned feature, the Fppro1 of high temprature detectation described in the present invention can be widely used in the industry such as washing, food, process hides.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE collection of illustrative plates of the recombinant expressed purifying of high temprature detectation gene Fppro1.In FIG, M is albumen Marker, swimming lane 1 is the expression of recombinant vectors pColdI-Fppro1 in E.coli BL21 (induction), swimming lane 2 is the target protein Fppro1(~ 60kDa through Ni-NTA purifying gained), swimming lane 3 is the abduction delivering of recombinant vectors pColdI-Fppro1 in E.coli BL21.
Fig. 2 is the optimum temperature of recombinase Fppro1.In fig. 2, X-coordinate is temperature (DEG C), and ordinate zou is the relative reactivity (%) of enzyme.
Fig. 3 is the thermostability of recombinase Fppro1.In figure 3, X-coordinate is soaking time (h), and ordinate zou is the relative activity (%) of enzyme; Respectively be labeled as: ■ 30 DEG C; ● 40 DEG C; ◆ 50 DEG C;
Fig. 4 is the Optimun pH of recombinase Fppro1.In the diagram, X-coordinate is pH value, and ordinate zou is the relative reactivity (%) of enzyme.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as Molecular Cloning: A Laboratory room handbook (New York:Cold Spring Harbor Laboratory Press, 2001) experiment condition described in, or according to the condition that reagent or instrument manufacturer facility business advise.
Production bacterial strain for the preparation of described high temprature detectation gene Fppro1 is Pacific Ocean heat color bacillus (FlammeovirgaPacifica) H2.This bacterial strain is preserved in China typical culture collection center, address on June 13rd, 2012: China. Wuhan. and Wuhan University, deposit number is: CCTCC NO:M2012229.
1. the preparation of high temprature detectation Fppro1
1) extraction of Flammeovirga Pacifica H2 genomic dna
With the Flammeovirga Pacifica H2 bacterial classification that transfering loop picking-80 DEG C is preserved, LB culture medium flat plate (a kind of bacteria culture medium, containing the Tryptones of 1%, the sodium-chlor of 1%, the yeast extract of 0.5%, the agarose of 1.5%) the single bacterium colony of upper line separation.37 DEG C of overnight incubation put by flat board after line, and picking single colony inoculation is to (a kind of bacteria culture medium, containing the Tryptones of 1%, the sodium-chlor of 1%, the yeast extract of 0.5%) in the LB nutrient solution pipe of 5mL, and 37 DEG C of wave and culture spend the night.Get the centrifugal 2min of culture 2000r/min of 1.5mL; Add 500 μ L6mol/L Guanidinium hydrochlorides again, mix gently, until cracking limpid (can consider and increase Guanidinium hydrochloride consumption); Add isopyknic phenol/chloroform/primary isoamyl alcohol, mixing, the centrifugal 4 ~ 5min of 15000r/min, moves into new pipe mutually by upper water; Slowly add equal-volume Virahol, after mixing, place 5min; The centrifugal 15s of 15000r/min, removes supernatant, and precipitation washes twice with 70% ethanol, dries up; Add 200 μ L TE solution (10mmol/L Tris-HCl, 1mmol/LEDTA), 2 μ L RNase A, 37 DEG C of enzymolysis 1h; Add 120 μ L Virahols, 2 μ L1mol/L MgCl
2, mix gently, leave standstill 10min; The centrifugal 5min of 15000r/min, removes supernatant, and precipitation washes twice with 70% ethanol, dries up; Be precipitated and dissolved in 50 μ L TE solution.
2) pcr amplification of high temprature detectation gene Fppro1 and sequential analysis
According to the gene annotation result after the order-checking of Flammeovirga Pacifica H2 strain gene group, design primers F: 5 '-CG
gGATCCaTG AAA AAT ACA ATT TCA-3 ' (being scribed ss BamH I restriction enzyme site) (SEQIDNo.3) and R:5 '-AC
aAGCTTcTA TTT TGA GAT CTT TTC-3 ' (being scribed ss Hind III digestion site) (SEQIDNo.4).With the Flammeovirga Pacifica H2 genomic dna extracted for template, the Fppro1 gene of the total length that increases with primers F and R.PCR reaction conditions is: contain in the reaction system of 50 μ L, 100ng template, 400nmo/L primers F, 400nmo/L primer R, 200 μm of o/L dNTP, 2.5mmo/L Mg
2+, 5U Primer Star HSTaq enzyme (purchased from TaKaRa company), 5mL10 × PCR reaction buffer; Reaction conditions: 94 DEG C of 5min denaturations; 98 DEG C of 10s, 55 DEG C of 45s, 72 DEG C of 2min circulation 30 circles; 72 DEG C extend 10min.Carry out enzyme with BamH I and Hind III after gained PCR primer purifying to cut, and connect with the pColdI carrier after BamH I and Hind III digestion with same, obtain pColdI-Fppro1 expression vector, adopt CaCl
2pColdI-Fppro1 is transformed in E.coli DH5 α by method, and picking positive colony checks order.
3) expression and purification of recombinase Fppro1
By step 2) in the correct recombinant expression vector pColdI-Fppro1 Transformed E .coli BL21 of sequence that obtains, choose positive colony 37 ° of C in containing the LB substratum of 100 μ g/mL penbritins and shake training to A
600when=0.6, add isopropylthio-β-D-galactoside (IPTG) to final concentration 100 μm of ol/L, after 16 ° of C induce 12h, bacterium liquid is collected in the centrifuge tube of 200mL, 5000 × g centrifugation bacterial cell.By bacterial cell Eddy diffusion, at the lysis buffer of 20mL, (lysis buffer formula is: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/L NaH
2pO
4, pH8.0) in, ultrasonication becomes translucent to bacterium liquid, the centrifugal 20min of 18000 × g, supernatant with mix with the Ni-NTA Agarose that lysis buffer balances in advance, 4 ° of C were in conjunction with 1 hour, and purge process illustrates according to purification kit (purchased from Qiagen company) carries out.The albumen of purifying through 12% SDS-PAGE electrophoretic analysis, its molecular weight is about 60kDa, and purity reaches more than 95% (result is see Fig. 1)
2. the embodiment of the basic characterization analysis of recombinase Fppro1
1) measuring method of proteinase activity
Take casein as substrate, adopt Forint phenol method to measure.Solution used comprises: forint uses solution (a commercially available folin solution mixes with two parts of water, shakes up), sodium carbonate solution (42.4g/L), trichoroacetic acid(TCA) (65.4g/L), gradient pH value damping fluid, casein solution (10.0g/L).Reaction process is as follows: in the little centrifuge tube of 1.5mL, add 800 μ L casein solution, puts into the modulated water-bath preheating 5min to temperature of reaction, adds 200 μ L enzyme liquid, slowly shakes up, put back in water-bath and react 10min.Add 270 μ L trichoroacetic acid(TCA)s, mix with termination reaction.
One control tube is set in addition, in control tube, adds 800 μ L casein solution, put into the modulated water-bath preheating 5min to temperature of reaction, add 200 μ L water, slowly shake up, put back in water-bath and react 10min.Add 270 μ L trichoroacetic acid(TCA)s and with the enzyme liquid after 100 DEG C of process 5min, mix.
By experiment tube and control tube with the centrifugal 2min of 13,000r/min, get 1mL supernatant liquor respectively in the empty test tube of corresponding numbering, add 5mL sodium carbonate solution afterwards more successively and 0.5mL forint uses solution, in 40 ° of C water-baths colour developing 20min after mixing.Measure the OD of each pipe
680value.And from ready-made Tyr typical curve, read the amount of the Tyr produced in reaction, calculate enzyme and live.
Protease activity unit of force (U) is defined as per minute caseinhydrolysate and produces enzyme amount needed for 1 μ g tyrosine.
2) the optimum temperature analysis of recombinase Fppro1
Adopt Tris-HCl damping fluid (pH8.0), the recombinase Fppro1 after purified is carried out enzymatic reaction and measure enzyme living at different temperatures.Live as 100% with the highest enzyme, calculate relative enzyme and live, draw temperature-enzyme curve (see figure 2) alive relatively.The optimum temperature of the recombinase Fppro1 that result shows in the present invention is 60 DEG C, and has wider operative temperature scope: the vigor that can keep more than 60 DEG C within the scope of 40 DEG C ~ 70 DEG C; The vigor of about more than 90% is kept within the scope of 50 DEG C ~ 65 DEG C; The vigor of about 40% can be kept 30 DEG C time.
3) thermal stability analysis of recombinase Fppro1
Adopt Tris-HCl damping fluid (pH8.0), by the recombinase Fppro1 held for some time at different temperatures after purified, then carry out enzymatic reaction, measure the vigor of residual protein enzyme, detect and react under optimal reactive temperature.Draw soaking time-relative activity curve (see figure 3).Result shows that the recombinase Fppro1 in the present invention has extraordinary thermostability.Be incubated 24h at 30 DEG C after, vigor is not lost; Enzyme activity residue more than 95% after 12h at 40 DEG C; After being incubated 1h at 50 DEG C, vigor has rising slightly on the contrary, can keep the vigor of about 50% after vigor keeps about 75%, 8h after can keeping 85%, 4h after 2h; Enzyme residue 40% alive after 60 DEG C of insulation 1h enzymes residues about 65%, 2h alive.
0.1mmol/L Ca is added in Tris-HCl damping fluid (pH8.0)
2+, then detecting the stability of recombinase Fppro1 at 60 DEG C (Fig. 3) by same steps, result shows Ca
2+significantly improve the thermostability of recombinase Fppro1, enzyme residue 76% alive after 60 DEG C of insulation 1h, after insulation 2h, residual enzyme work is 65%, all than not having Ca
2+time 65% and 40% high.
So far research report and application/authorize patent in, if do not add stablizer, the thermostability of initial proteolytic enzyme more than 40 DEG C is all poor, best a kind of thermostable proteinase produced by bacillus thuringiensis being Chinese patent ZL200610069339.2 and providing, its optimum temperature is 55 DEG C, the basic non-inactivation of 1h under 55 DEG C of conditions, under 60 DEG C of conditions, 1h residual activity is 60%.Therefore the recombinase Fppro1 in the present invention has very outstanding thermostability.
4) the suitableeest action pH analysis of recombinase Fppro1
Purified recombinase Fppro1, in the damping fluid of different pH value, carries out reacting to measure its optimal pH at 60 DEG C, lives as 100% with the highest enzyme, calculates relative enzyme and lives, and draws the relative enzyme of pH-curve (see figure 4) alive.The Optimun pH of the recombinase Fppro1 that result shows in the present invention is 9, can keep the vigor of more than 80% in the scope of pH8 ~ 10; The vigor of about 65% can be kept when pH7, there is wider pH sphere of action.
Claims (8)
1. a high temprature detectation, it is characterized in that described high temprature detectation called after Fppro1, it produces bacterial strain is Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2, this bacterial strain is preserved in China typical culture collection center on June 13rd, 2012, address: China. Wuhan. Wuhan University, deposit number is: CCTCC NO:M2012229;
The sequence of described high temprature detectation Fppro1 encoding gene is as follows:
2. a kind of high temprature detectation as claimed in claim 1, is characterized in that the aminoacid sequence of high temprature detectation Fppro1 is as follows:
3. the preparation method of high temprature detectation Fppro1, is characterized in that comprising the following steps:
1) high temprature detectation Fppro1 encoding gene is as claimed in claim 1 cloned;
2) by step 1) clone the high temprature detectation Fppro1 encoding gene insertion expression vector obtained, build the recombinant expression vector carrying described high temprature detectation Fppro1 encoding gene;
3) recombinant expression vector is transformed in intestinal bacteria (E.coli) BL21;
4) choose the positive colony transformed in rear E.coli BL21 and carry out fermentation culture in substratum;
5) the E.coli BL21 cell after collected by centrifugation fermentation, resuspended described E.coli BL21 cell carries out cracking in lysis buffer;
6) by step 5) in suspension after cracking centrifugal, collect supernatant liquor, then mix with Ni-NTA Agarose, carry out purifying according to purification kit specification sheets, obtain high temprature detectation Fppro1.
4. the preparation method of high temprature detectation Fppro1 as claimed in claim 3, is characterized in that in step 2) in, described expression vector is selected from pCold I carrier.
5. the preparation method of high temprature detectation Fppro1 as claimed in claim 3, is characterized in that in step 4) in, described substratum is selected from the LB substratum containing 100 μ g/mL penbritins.
6. the preparation method of high temprature detectation Fppro1 as claimed in claim 3, is characterized in that in step 4) in, the condition of described fermentation culture is: temperature is 37 DEG C, and shaking table is cultured to A
600when=0.6, then to add isopropylthio-β-D-galactoside to final concentration be 50 μm of ol/L, under 16 DEG C of conditions, induce 12h.
7. the preparation method of high temprature detectation Fppro1 as claimed in claim 3, is characterized in that in step 5) in, described lysis buffer formula is: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/L NaH
2pO
4, pH 8.0.
8. the preparation method of high temprature detectation Fppro1 as claimed in claim 3, is characterized in that in step 6) in, described centrifugal condition is 18000 × g, centrifugal 20min, 4 DEG C.
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