CN101805719A - Method for expression of streptoverticillium mobaraense transglutaminase and dedicated engineering bacteria thereof - Google Patents
Method for expression of streptoverticillium mobaraense transglutaminase and dedicated engineering bacteria thereof Download PDFInfo
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Abstract
The invention discloses a method for the expression of streptoverticillium mobaraense (microbial) transglutaminase (MTG) and dedicated engineering bacteria thereof. The dedicated engineering bacterias are obtained by introducing the expression vector containing the genes of streptoverticillium mobaraense transglutaminase with the GenBank accession number being AF531437 into escherichia coli. The method of the invention comprises the following steps: fermenting the dedicated expression engineering bacteria of the streptoverticillium mobaraense transglutaminase; collecting inclusion bodies; and subjecting the inclusion bodies to rinsing, denaturation, renaturation and purification to obtain the streptoverticillium mobaraense transglutaminase. The invention provides an effective method for the high-efficiency and low-cost mass-production of transglutaminase, thus ensuring higher application value.
Description
Technical field
The present invention relates to relate to the expression method and the dedicated engineering bacteria thereof of Streptomyces mobaraensis Transglutaminase EC2.3.2.13.
Background technology
(Microbial Transglutaminase, MTG are that the γ-acyl group of glutamine in a kind of catalytic polypeptide chain is transferred to the amino in another substrate E2.3.2.13) to the glutamine of microbe transaminase, form the transferring enzyme of gamma-glutamyl compound.As substrate is protein, the crosslinking reaction between two protein of its energy catalysis.Substrate comprises (Zhu Y such as whey-protein, wheat germ protein, soybean protein, ox myosin and poultry actomyosin, Rinzema A, .TramperJ, Bol J.Microbal transglutaminase-a review of its production and applicationin food processing.Appl.Microbiol.Biotechnol, 1995.44:277-282).By the crosslinking reaction between catalytic proteins or the polypeptide, can improve functions such as proteinic water-soluble, whipability, agitatability, thermostability, emulsifying property, thereby the aspects such as matter structure, outward appearance and local flavor of protein-based food are improved, and improve proteinic commercial value.At present, MTG has been widely used in foodstuffs industry (Ye DY (Ye Danying), Peng ZY (Peng Zhiying), Zhao MM (Zhao Mouming), Transglutaminase and its application in foodprocessing.Journal of Zhongzhou Grain College (Zhengzhou Grain College journal), 2000.21 (2): 46-49).Transglutaminase extensively is present in the multiple animal tissues, but owing to low, the separation difficulty of content, poor heat stability, calcium ion had factor such as dependency, difficult it is carried out suitability for industrialized production.The end of the eighties in last century, discovery Streptomyces mobaraensis such as Ando, streptoverticillum and grey meat streptoverticillium etc. all can be secreted MTG, and the excretory MTG of these Institute of Micro-biology has the thermostability height, does not need calcium ion, characteristics (Ando H such as product crosslinking degree height, Adachi M, Umeda K, et al.Purification and characteristics of a noveltransgloutaminase derived from microorganism.Agric Biol.Chem, 1989.53:2613-2617.).Though MTG now can carry out suitability for industrialized production, production cost is higher, has limited its further application.
Summary of the invention
The expression method and the dedicated engineering bacteria thereof that the purpose of this invention is to provide a kind of Streptomyces mobaraensis Transglutaminase EC2.3.2.13.
The dedicated engineering bacteria of expression Streptomyces mobaraensis Transglutaminase EC2.3.2.13 provided by the present invention obtains containing in the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier importing intestinal bacteria; GenBank number of described Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene is AF531437.
Be used to make up the described carrier that sets out that contains Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier and can be pET-22b, pET-28 or pET-15 etc., be preferably pET-22b.
Be the carrier that sets out with pET-22b, the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier that contains of structure is pET-MTG.
Described intestinal bacteria are the coli strain that is suitable for protein expression, are preferably E.coli Rosetta (DE3).
The method that will contain Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier transformed into escherichia coli can be method for transformation commonly used in the bioengineering field, as the protoplast transformation method of heat shock method, electrotransformation, joint conversion method, PEG mediation etc.
Second purpose of the present invention provides a kind of expression method of Streptomyces mobaraensis Transglutaminase EC2.3.2.13.
The expression method of Streptomyces mobaraensis Transglutaminase EC2.3.2.13 provided by the present invention, it is the dedicated expression engineered bacteria of the above-mentioned Streptomyces mobaraensis Transglutaminase EC2.3.2.13 of fermentation, the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 of expressing is present in the somatic cells with the form of inclusion body, collect inclusion body, it is carried out obtaining the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 behind purifying, sex change, renaturation and the purifying.
Need add the IPTG inductor when fermenting above-mentioned engineering bacteria, add IPTG concentration be 0.8-1.2mmol/L, be preferably 1mmol/L, inducing temperature is 35-39 ℃, is preferably 37 ℃, induction time is 2-4 hour, is preferably 3 hours.
Inclusion body is carried out purifying be may further comprise the steps: inclusion body is resuspended with the Tris-HCl damping fluid that contains Triton.
Can may further comprise the steps inclusion body sex change, renaturation: inclusion body is being contained 8mol/L urea, and the Tris-HCl of 20mmol/L DTT and 1mmol/L EDTA after the dissolving, regulates pH value to 4.0 rapidly in the pH6.5-9.0 damping fluid, causes the MTG renaturation.
The concrete method that inclusion body is carried out sex change, renaturation can be: the inclusion body through washing will be dissolved in 20mol, pH6.5-9.0, and contain 8mol/L urea, in the Tris-HCl damping fluid of 20mmol/L DTT and 1mmol/L EDTA, stirring at room 2 hours, centrifugal removal post precipitation, adjust pH to 4.0, recentrifuge is removed precipitation, with supernatant liquor with after 50 times of the acetate buffer of 20mmol, pH 4.0 dilutions 4 ℃ stirred 12-24 hour, adjust pH to 6.0, centrifugal removal post precipitation is dialysed 1-3 time with the phosphoric acid buffer (PB) of 10 times of 20mmol, pH6.0.
The method of renaturation product being carried out purifying can be strong cation exchange chromatography, weak cation exchange chromatography etc., is preferably the strong cation exchange chromatography.
The invention provides a kind of protokaryon high-efficiency expression method of Streptomyces mobaraensis Transglutaminase EC2.3.2.13.This method is that the Transglutaminase EC2.3.2.13 gene of Streptomyces mobaraensis is imported in the intestinal bacteria by coli expression carrier, obtains the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 through fermentation.Wherein p-ET22b is preferred coli expression carrier, and this carrier contains the encoding sequence of pelB signal peptide, and after use restriction enzyme Nde I excised this sequence, expression product did not just contain signal peptide.(Kikuchi Y such as the Transglutaminase EC2.3.2.13 gene order of the Streptomyces mobaraensis of being cloned and Kikuchi, Date M, Yokoyama K, Umezawa Y, Matsui H.Secretion of active-formStreptoverticillium mobaraense transglutaminase by Corynebacteriumglutamicum:processing of the pro-transglutaminase by a cosecretedsubtilisin-Like protease from Streptomyces albogriseolus.Appl Environ.Microbiol, 2003,69:358-66.) report the Transglutaminase EC2.3.2.13 gene order identical, G+C content is 66.66%, and contain a large amount of rare codons, wherein AGA and AGG account for 37.8% (11/29) in the coding Arg codon, show that the synthetic needs of Transglutaminase EC2.3.2.13 have the tRNA of rare codon
Arg (AGG/AGA)But contain among the tRNA of rare codon tRNA all intestinal bacteria
Arg (AGG/AGA)Content is minimum, therefore when using E.ColiBL-21 (DE3) and E.Coli BL-21 (DE3) PLys as the host bacterium, does not see the expression band of reorganization Transglutaminase EC2.3.2.13 after IPTG induces, and may be the tRNA of rare codon in this two strain host bacterium
Arg (AGG/AGA)Contain due to the quantity not sufficient.And after using E.Coli Rosetta (DE3) the host bacterium of energy coexpression rare codon, a large amount of tRNA
Arg (AGG/AGA)Gene co-expressing makes the reorganization Transglutaminase EC2.3.2.13 obtain to efficiently express.Experimental results show that through shake-flask culture to obtain wet thallus 5.8g/L substratum, obtain the inclusion body 0.55g of purifying, the final pure enzyme 102.4mg of reorganization Transglutaminase EC2.3.2.13 that obtains; If use fermentation process in high density, every liter of substratum can obtain to surpass the 100g wet thallus, finally obtain the pure enzyme of 2g, far above existing 143mg natural enzyme/L substratum (can) expression level (Yokoyama KI, NakamuraN, Seguro K, Kubota K.Overproduction of microbial transglutaminase inEscherichia coli, in vitro refolding, and characterization of the refoldedform.Biosci Biotechnol Biochem.2000,64:1263-70.).The present invention has higher actual application value for efficient, a large amount of, low cost production Transglutaminase EC2.3.2.13 provide a kind of effective ways.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 is 1% an agarose gel electrophoresis detected result of the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene of pcr amplification
1% agarose gel electrophoresis detected result of the Nde I of the positive cloned plasmids of Fig. 2 and Xho I double digestion product
Fig. 3 is the 15%SDS-PAGE detected result of inducing the whole bacterial protein of three kinds of forward and backward reorganization bacterium through IPTG
Fig. 4 is the 15%SDS-PAGE detected result of purified reorganization Streptomyces mobaraensis Transglutaminase EC2.3.2.13
Fig. 5 is the MTG behind the SP-Sepharose FF ion exchange chromatography purification renaturation
Fig. 6 is for detecting the experimental result of reorganization Streptomyces mobaraensis Transglutaminase EC2.3.2.13 to the bovine serum albumin crosslinked action
Fig. 7 produces curve for the thalline of reorganization bacterium MTG/Rosetta (DE3)
Fig. 8 is the SDS-PAGE detected result of the high density fermentation product of reorganization bacterium MTG/Rosetta (DE3)
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and all percentage concentrations are mass percent concentration.
Experiment material:
Colibacillus BL21 (DE3) PLysS, Rosetta (DE3) and plasmid pET-22b are available from Novagen company; Restriction enzyme Nde I, Xho I and T
4Dna ligase is given birth to the worker available from Dalian TaKaRa company, high-fidelity Pfu polysaccharase available from Shanghai; Plasmid extracts test kit on a small quantity, gel reclaims test kit available from Shanghai Hua Shun company; Primer synthesizes and dna sequencing work is finished by Shanghai Bo Ya company; SP-Sepharose is available from Amersham company; The molecular weight of albumen standard is available from Gibco company; Other reagent such as IPTG, urea, EDTA are import or homemade analytical reagent.
The acquisition of embodiment 1, Streptomyces mobaraensis Transglutaminase EC2.3.2.13
One, the structure of the dedicated expression engineered bacteria of Streptomyces mobaraensis Transglutaminase EC2.3.2.13
1, the clone of Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene
According to known Streptomyces mobaraensis Transglutaminase EC2.3.2.13 (MTG) gene (GenBank number: AF531437) open reading frame design primer, primer sequence is as follows:
P1 (forward primer): 5 '-TAAAAACATATGGACTCCGACGACAGGGTCAC-3 ';
P2 (reverse primer): 5 '-TAAAAACTCAGGTTACGGCCAGCCCTGCTTTACC-3 '
Genomic dna with 1 μ g Streptomyces mobaraensis (Streptoverticillium mobaraense) is a template, under the guiding of primer P1 and P2, and pcr amplification Transglutaminase EC2.3.2.13 gene.100 μ l PCR reaction systems are:
10 * amplification buffer 10ul,
4 kinds of each 200umol/L of dNTP mixture,
Each 10～100pmol of primer,
Template DNA 0.1～2ug,
Taq archaeal dna polymerase 2.5u,
Mg
2+ 1.5mmol/L，
Add two or tri-distilled water to 100ul.
Reaction conditions is: 94 ℃ of 5min at first; 94 ℃ of 50s then, 58 ℃ of 1.5min, 72 ℃ of 2min, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, detected result is (swimming lane Marker is DNA marker (bp), and swimming lane MTG is a pcr amplification product) as shown in Figure 1, pcr amplification has gone out the dna fragmentation of 993bp as a result, conforms to expected results.
2, the structure that contains the coli expression carrier of Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene
Reclaim with gel that test kit reclaims and the dna fragmentation of the 993bp of purification step 1 usefulness PCR method amplification, to its with restriction enzyme Nde I, Xho I double digestion after, with carrier pET-22b T through same enzyme double digestion
4DNA couples together, behind 16 ℃ of reaction 16h, to connect product and transform colibacillus DH5 α competent cell, after screening, select positive colony, the upgrading grain carries out the double digestion analysis verification with restriction enzyme Nde I, Xho I, enzyme is cut product carry out the detection of 1% agarose gel electrophoresis, (swimming lane 1 is DNA marker (bp) to detected result as shown in Figure 2, swimming lane 2 is pET-MTG/Nde I+Xho I), the result has obtained the endonuclease bamhi of 993bp, shows that the purpose fragment correctly is cloned among the carrier pET-22b.Identify that to cutting correct positive colony plasmid carries out dna sequencing and identifies then through enzyme, sequencing result shows the nucleotide sequence that inserts dna fragmentation, and (GenBank number: nucleotide sequence AF531437) is consistent with known MTG gene, the coli expression carrier that contains Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene that shows structure is correct, with its called after pET-MTG.
3, transform
The coli expression carrier pET-MTG of the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene that step 2 is made up transforms colibacillus Rosetta (DE3), BL21 (DE3) PlysS and E.coli BL21 (DE3) bacterial strain respectively, the two kinds of bacterial strains in back are contrast, obtain the dedicated expression engineered bacteria of Streptomyces mobaraensis Transglutaminase EC2.3.2.13.With above-mentioned three kinds of reorganization bacterium difference called after MTG/Rosetta (DE3), MTG/BL21 (DE3) PlysS and MTG/BL21 (DE3).
Those skilled in the art all can obtain the dedicated expression engineered bacteria MTG/Rosetta (DE3) of Streptomyces mobaraensis Transglutaminase EC2.3.2.13 by above step operation, and this reorganization bacterium has high performance reproducibility.The construction process of reorganization bacterium is seen non-patent literature Francois Baneyx.Recombinant protein expression in Escherichiacoli.Current Opinion in Biotechnology.1999.10:411-421., and the applicant promises to undertake that the public can obtain this reorganization bacterium MTG/Rosetta (DE3) from the applicant.
The purifying of sex change, renaturation and the MTG of two, the expression of MTG in colibacillus, inclusion body
Single bacterium colony of the reorganization bacterium MTG/BL21 (DE3) that the picking step 1 obtains, it is inoculated in the LB liquid nutrient medium that contains the 50mg/mL acillin, single bacterium colony of back two kinds of reorganization bacterium MTG/Rosetta (DE3) and MTG/BL21 (DE3) PlysS is inoculated in respectively in the LB liquid nutrient medium that contains 50mg/mL acillin and 30mg/mL paraxin, 37 ℃ of shaking culture are when the OD value is 0.6-0.8, adding final concentration is the IPTG of 1mmol/L, induces 3 hours for 37 ℃.After inducing end, the whole bacterial protein of inducing three kinds of forward and backward reorganization bacterium through IPTG is carried out 15%SDS-PAGE to be detected, detected result is (swimming lane 1, Molecular weight marker (KD) as shown in Figure 3, before swimming lane 2-4 IPTG induces: swimming lane 2, E.coli BL 21 (DE3), swimming lane 3, E.coli BL 21 (DE3) PlysS, swimming lane 4, E.coli Rosetta (DE3); Swimming lane 5-7:IPTG induces the back: swimming lane 5, E.coli BL21 (DE3), swimming lane 6, E.coli BL21 (DE3) PlysS, swimming lane 7, E.coli Rosetta (D3E)), have only through IPTG inductive MTG/Rosetta (DE3) and the exogenous protein expression band that molecular weight is 36kDa occurs, consistent with the molecular weight of MTG, show in the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene intestinal bacteria to obtain to express, and MTG/BL21 (DE3) PlysS, MTG/BL21 (DE3) and the exogenous protein expression band all do not occur without IPTG inductive recombinant bacterial strain.
Centrifugal collection obtains wet thallus 5.8g/L substratum through IPTG inductive MTG/Rosetta (DE3) thalline.With 50mmol/L, pH 8.0 Tris-HCl (containing 1mmol/L EDTA) damping fluid suspension thalline, ultrasonic disruption thalline in ice bath, collect respectively after centrifugal and go up cleer and peaceful precipitation, to precipitate part (inclusion body) 50m molTris-HCl (pH8.0, contain 100m mol/L NaCl, 1m mol/L EDTA, 0.5%Triton X-100) damping fluid is resuspended, after supersound process washing 3 times, again with the same buffer washing that does not contain Triton X-100 3 times, obtain the inclusion body of purifying, weight in wet base 0.55g.Inclusion body (protein concentration is about 20mg/mL) with purifying is dissolved in 20mol then, pH8.0 Tris-HCl (contains 8mol/L urea, 20m mol/L DTT and 1m mol/L EDTA) carry out sex change in the damping fluid, stirring at room 2 hours, centrifugal removal post precipitation, with HCl adjust pH to 4.0, recentrifuge is removed precipitation, and with 50 times of the acetate buffer dilutions of 20m mol, pH4.0,4 ℃ were stirred 12-24 hour with supernatant liquor.With 0.5mol NaOH adjust pH to 6.0, centrifugal removal precipitation is dialysed supernatant liquor 2 times with 20m mol, the pH6.0 phosphoric acid buffer (PB) of 10 times of volumes.With SP-Sepharose strong cat ion exchange column chromatography purification on the renaturation product, with the unconjugated composition of PB flush away, with containing 0.5M NaCl eluant solution, elutriant carries out ultrafiltration and concentration after dialysis again, obtains MTG protein 10 2.4mg at last.Carry out the 15%SDS-PAGE detection with concentrating the reorganization MTG protein sample that obtains, (swimming lane 1 is the molecular weight of albumen standard to detected result as shown in Figure 4, swimming lane 2 is an inclusion body, swimming lane 3 is for being purified reorganization MTG albumen), MTG behind the SP-Sepharose FF ion exchange chromatography purification renaturation is (Sample (1): Refolded MTG in PB as shown in Figure 5, EluentA (2): PB+20m mol/L NaCl, EluentB (3): PB+500m mol/L NaCl), show to have obtained highly purified Streptomyces mobaraensis Transglutaminase EC2.3.2.13.
The determination of activity of embodiment 2, Streptomyces mobaraensis Transglutaminase EC2.3.2.13 and to the crosslinked action of bovine serum albumin (BSA)
One, the determination of activity of Streptomyces mobaraensis Transglutaminase EC2.3.2.13
Express also activity (the Pasternack R of the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 of purifying with colorimetric method for determining embodiment 1, Dorsch S, Otterbach J, Robenek Is, et al.Bacterialpro-transglutaminase from Streptoverticillium mobaraense.Purification, characterization and sequence of the zymogen.Eur J Biochem, 1998.257:570-576).The enzyme of MTG unit alive is: in the time of 37 ℃, catalytic substrate CBZ-Gln-Gly generates 1 μ mol L-glutamy γ-different hydroxyoxime acid of list.The different hydroxyoxime acid of L-glutamy γ-list has absorption peak at wavelength 520nm place, the ratio of the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 that the result is expressed is lived and is 21.5u/mg, compare with the natural enzyme activity (ratio of natural enzyme is lived and is 23u/mg) of bibliographical information, the ratio of reorganization MTG low slightly (Kikuchi Y alive, Date M, Yokoyama K, Umezawa Y, Matsui H.Secretion of active-form Streptoverticilliummobaraense transglutaminase by Corynebacterium glutamicum:processing of thepro-transglutaminase by a cosecreted subtilisin-Like protease fromStreptomyces albogriseolus.Appl Environ.Microbiol, 2003,69:358-66.).
Two, detect of the crosslinked action of Streptomyces mobaraensis Transglutaminase EC2.3.2.13 to bovine serum albumin (BSA)
BSA is dissolved in the phosphoric acid buffer-physiological saline (PBS) that contains 2mmol DTT, making protein concentration is 1mg/mL, the reaction cumulative volume is 1mL, earlier at 50 ℃ of insulation 10min, add 1u embodiment 1 then and express the also Streptomyces mobaraensis Transglutaminase EC2.3.2.13 of purifying, 37 ℃ of insulations, control group is the reaction system that does not add reorganization MTG, in 10,20,30 and 60min sampling carry out 10%SDS-PAGE, detect the crosslinked situation of BSA.
The result as shown in Figure 6, precipitation appears in BSA after handling with DTT, the result is with MTG the no tangible polymer appearance of group ( swimming lane 1,6～10); Reorganization MTG treatment group (swimming lane 2～5) has formed the bigger polymer of molecular weight, because molecular weight is too big, polymer is failed to enter concentrated glue (the aperture ratio separation gel that concentrates glue is little) and is attached to concentrated glue surface, sample hole, shows that the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 has stronger crosslinked action to bovine serum albumin.
The reorganization bacterium MTG/Rosetta (DE3) that expresses highly purified Streptomyces mobaraensis Transglutaminase EC2.3.2.13 is carried out high density fermentation.The OD value curve of reorganization thalli growth is (PO as shown in Figure 7
2: dissolved oxygen .OD: cell density .Hour: fermentation time (hour)), showing that the OD value began to rise in inoculation in 2 hours, lift velocity is accelerated after 3 hours, and lift velocity slows down after 26 hours, and the OD value of this moment is 56.The dissolved oxygen curve display: dissolved oxygen begins to descend behind the inoculation 4h, and straight line descends after 6 hours.Dissolved oxygen drops to 28% behind the 8h, and the alkali pump discontinuous running shows bacterium producing acid simultaneously, and about 10 hours, dissolved oxygen began to rise, and illustrates that nutriment consumes gradually in the fermentor tank, begins feed supplement when 15h.When the OD value for the treatment of cell density reaches 60-65, stop feed supplement half an hour, allow glucose fully exhaust.IPTG induces back MTG expression to increase gradually, reaches the highest at 3 hours, and expression amount decline after 4 hours (as shown in Figure 8, swimming lane 1.Marker, before swimming lane 2.IPTG induced, swimming lane 3-6.IPTG induced back 1h, 2h, 3h, 4h.).When inducing 3h, express a band band close and merge with it, can't judge that MTG accounts for the percentage composition of whole bacterial protein.In high density fermentation subsequently, determine that induction time is 3 hours, the centrifugal wet thallus 105g/L substratum that gets.Separation and purification inclusion body as stated above, and carry out the renaturation purifying, obtain MTG albumen 1.85g altogether.
More than describe and can sum up outstanding feature of the present invention:
1, pET22b is secreted expression carrier, contains the coded sequence of pelB signal peptide, and the present invention uses the NdeI enzyme to cut and dispels this sequence, and therefore, expression product does not contain signal peptide. It is identical that the sequence dna fragment that recombinant plasmid pET-MTG inserts and Kikuchi etc. report, wherein G+C accounts for 66.66%. When external source genes of interest during at Bacillus coli expression, because the preferences difference of codon, can in default of certain and several tRNA, directly cause translation to be ended or mistake. Coding arginine codon AGA, AGG, codon glycine GGA and proline CCC seldom are used. Contain among the tRNA of rare codon tRNA all Escherichia coliarg(AGG/AGA)Content is minimum. The analysis of MTG encoding gene shows: contain Escherichia coli rare codon AGA, AGG, CCC, GGA, wherein AGA and AGG are maximum, account for 37.8% (11/29) in the coding Arg codon. Be positioned at the Arg that MTG N-holds the 5th and 15, codon is AGG., more is unfavorable for the expression of MTG. The reports such as Ivanov contain the rare codon period of the day from 11 p.m. to 1 a.m when mRNA5 holds, and this gene obviously is suppressed at Bacillus coli expression. The present invention studies show that: when using E.coli BL-21 (DE3) and BL-21 (DE3) PLysS as Host Strains, do not see after IPTG induces and express band that this may be the tRNA that contains rare codon in this two strains Host Strainsarg(AGG/AGA)Contain due to the quantity not sufficient. And the used special-purpose recombinant bacterium Rosetta of the present invention (DE3) is through modifying, being exclusively used in the bacterial strain with the exogenous protein expression of Escherichia coli rare codon, so make MTG realize efficiently expressing.
2, in the renaturing inclusion bodies process, the present invention has used dilution refolding, regulates rapidly pH value to 4.0, and this technology is simple and amplify easily suitable suitability for industrialized production. The present invention adds Triton in the inclusion body purification renaturation process, make renaturation yield improve 40%. Use fermentation process in high density, every liter of culture medium obtains the 105g wet thallus, has obtained the pure enzyme of nearly 1.85g, far above the highest expression (every liter of culture medium of 132mg/) of present report. Activity test shows: compare the present invention's specific activity of MTG almost identical (specific activity of restructuring MTG is 21.5u/mg, and the specific activity of native enzyme is 23u/mg) of recombinating with bibliographical information native enzyme activity.
In a word, the present invention has realized the High level prokaryotic expression of MTG, and has set up high cell density fermentation and simple and effective renaturing inclusion bodies method, provides assurance for further utilizing the more cheap microbial transglutaminase of Escherichia coli suitability for industrialized production.
Claims (10)
1. express the dedicated engineering bacteria of Streptomyces mobaraensis Transglutaminase EC2.3.2.13, obtain containing in the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier importing intestinal bacteria; GenBank number of described Streptomyces mobaraensis Transglutaminase EC2.3.2.13 gene is AF531437.
2. dedicated engineering bacteria according to claim 1 is characterized in that: making up the described carrier that sets out that contains Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier is pET-22b, pET-28 or pET-15 etc., is preferably pET-22b.
3. dedicated engineering bacteria according to claim 2 is characterized in that: being used to make up the described carrier that sets out that contains Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier is pET-22b.
4. dedicated engineering bacteria according to claim 3 is characterized in that: the described Streptomyces mobaraensis Transglutaminase EC2.3.2.13 expression carrier that contains is pET-MTG.
5. dedicated engineering bacteria according to claim 1 is characterized in that: described intestinal bacteria are E.coliRosetta (DE3).
6. the expression method of a Streptomyces mobaraensis Transglutaminase EC2.3.2.13, it is the dedicated expression engineered bacteria of the described Streptomyces mobaraensis Transglutaminase EC2.3.2.13 of fermentation claim 1, collect inclusion body, it is carried out obtaining the Streptomyces mobaraensis Transglutaminase EC2.3.2.13 after purifying, sex change, the renaturation.
7. expression method according to claim 6 is characterized in that: need add the IPTG inductor during described fermentation, add IPTG concentration be 0.8-1.2mmol/L, inducing temperature is 35-39 ℃, induction time is 2-4 hour.
8. according to claim 6 or 7 described expression methods, it is characterized in that: can may further comprise the steps: inclusion body is resuspended with the Tris-HCl damping fluid that contains Triton inclusion body purification; Can may further comprise the steps inclusion body sex change, renaturation: inclusion body contains 8mol/L urea at pH6.5-9.0, after the dissolving, regulates pH value to 4.0 rapidly in the Tris-HCl damping fluid of 20mmol/L DTT and 1mmol/L EDTA, causes the MTG renaturation.
9. expression method according to claim 8, it is characterized in that: the described detailed process that inclusion body is carried out sex change, renaturation is: the inclusion body through washing is dissolved in the described Tris-HCl damping fluid of 20mol, stirring at room 2 hours, centrifugal removal post precipitation, adjust pH to 4.0, recentrifuge is removed precipitation, with supernatant liquor with after 50 times of the acetate buffer of 20mmol, pH 4.0 dilutions 4 ℃ stirred 12-24 hour, adjust pH to 6.0, centrifugal removal post precipitation is dialysed 1-3 time with the phosphoric acid buffer of 10 times of 20mmol, pH6.0.
10. according to claim 6 or 7 or 8 or 9 described expression methods, it is characterized in that: the described method that renaturation product is carried out purifying is the strong cation exchange chromatography.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102517242A (en) * | 2011-12-30 | 2012-06-27 | 齐河百多安生物医药科技有限公司 | Method for improving transglutaminase protein expression index of streptoverticillium mobaraense and dedicated engineering bacterium thereof |
| CN106148296A (en) * | 2016-09-30 | 2016-11-23 | 南京工业大学 | A kind of production method of glutamine transaminage of recombinating |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1796561A (en) * | 2004-12-20 | 2006-07-05 | 南京大学 | Recombined aminotransierase gene of glutamine of microbe, and preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1796561A (en) * | 2004-12-20 | 2006-07-05 | 南京大学 | Recombined aminotransierase gene of glutamine of microbe, and preparation method |
Non-Patent Citations (1)
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| 《生 物 工 程 学 报》 20050930 徐斌，等 茂原链轮丝菌转谷氨酰胺酶基因在大肠杆菌中高效表达 第794－798 1－10 第21卷, 第5期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102517242A (en) * | 2011-12-30 | 2012-06-27 | 齐河百多安生物医药科技有限公司 | Method for improving transglutaminase protein expression index of streptoverticillium mobaraense and dedicated engineering bacterium thereof |
| CN106148296A (en) * | 2016-09-30 | 2016-11-23 | 南京工业大学 | A kind of production method of glutamine transaminage of recombinating |
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