CN101955960A - Method for purifying recombinant lactase by using elastin like protein tag - Google Patents
Method for purifying recombinant lactase by using elastin like protein tag Download PDFInfo
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- 108010059881 Lactase Proteins 0.000 title claims abstract description 22
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for purifying recombinant lactase by using an elastin like protein tag. The method comprises the following steps of: (1) connecting a lactase gene tt412 and an elastin like protein tag ELP [V5-80] to a prokaryotic expression vector pET-32a(+) sequentially so as to construct a lactase fusion expression vector pET32a-tt412-ELP (V5-80); (2) performing electroporation transformation on an escherichia coli expression strain BLR (DE3) by using the constructed expression vector and performing self-induction expression on an identified positive transformant for 50 to 80 hours; (3) collecting the strain, grinding the strain with ultrasonic waves, centrifugally removing insoluble substances to obtain crude lactase enzyme liquid and standing the crude lactase enzyme liquid in a refrigerator of which the temperature is 4 DEG C for later use; (4) determining the phase transition temperature of a crude lactase enzyme liquid precipitate; and (5) purifying lactase recombinant protein by an inverse transition cycling (ITC) method. The method of the invention has the advantages of simpleness, rapidness, high efficiency, usability for large-scale separation and purification of lactase and other industrial enzyme preparations and high practical application value in the production of an enzyme preparation.
Description
Technical field
The present invention relates to a kind of purification process of enzyme, relate in particular to a kind of method of utilizing class elastin label by non-chromatographic process purification of Recombinant Sumylact L, belong to biological technical field.
Background technology
Sumylact L (EC:3.2.1.23) also claim beta-galactosidase enzymes, it can be decomposed into lactose the glucose and the semi-lactosi of easy absorption and sweet taste quality better, and in the hydrolysis sugar process, can also change the glucosides reaction, generation has the oligomeric galactose of different physiological roles, therefore it is the key that solves the lactose intolerance problem, promptly by developing low-lactose dairy product or directly allowing the oral Sumylact L of patient treat lactose intolerance.Yet Sumylact L is used seldom in the dairy industry of China so far, a major reason is that the most Sumylact Ls of present China are still dependence on import, and the Sumylact L of import costs an arm and a leg (about 1,000,000 yuans per ton), makes the use of Sumylact L be very restricted.Cause the high reason of Sumylact L price to mainly contain 2 points: the first, bacterial strain (as Kluyveromyces fragilis, Kluyveromyces lactis, aspergillus niger and the aspergillus oryzae) fermentation period of producing Sumylact L is long, and output is lower; The second, the Sumylact L that a lot of Institute of Micro-biology produce belongs to intracellular enzyme, needs the separation and purification of process multistep just can obtain all higher lactase formulation of purity and enzyme activity.Since these purification procedures consuming time many, efficient is low, material requested costs an arm and a leg, thereby makes the cost of separation and purification occupy the production cost of Sumylact L 60%-70%.The way that solves above-mentioned first problem normally utilizes modern genetic engineering and fermentation engineering to produce Sumylact L, and its advantage is the output height, and production cost is low, and the cycle is short, and is not subjected to the influence of factors such as season, geographical position.Since the seventies in 20th century, intestinal bacteria are the most deep, the most perfect, the most widely used expression systems of development of research in the genetically engineered always, and culture condition is simple, growth and breeding is fast because intestinal bacteria have, security is good, can efficiently express characteristics such as different exogenous genes products.A large amount of valuable polypeptide and protein has obtained overexpression in intestinal bacteria at present.Therefore adopt escherichia expression system production reorganization Sumylact L, can in the short production cycle, obtain a large amount of reorganization Sumylact Ls, thereby reduce the production cost of Sumylact L to a certain extent.Yet the Sumylact L that utilizes escherichia expression system production also is an intracellular enzyme, and its separation and purification also needs through treatment steps such as lysis, affinity chromatographys.Though affinity chromatography wherein has characteristics such as high selection, the high reactivity rate of recovery and high purity, and become one of otherwise effective technique of biomacromolecules such as present protein purification, but its shortcoming also clearly, mainly comprise consuming time big, cost is high, treatment capacity is little etc., these defectives make that not only the production technique cost of this protein purification is higher, and have limited its large-scale industrialization and used.Understand according to us, utilize escherichia expression system production reorganization Sumylact L at present both at home and abroad, what its separation purifying technique still adopted basically is the method that affinity chromatography is carried out in lysis then.
Elastin (elastin) belongs to extracellular matrix protein, and (extracellular matrix ECM), mainly is distributed in histoorgans such as blood vessel, skin, ligament and lung.Elastin can provide enough elasticity and restorer, thereby makes histoorgan stand the distortion of repetition and reversible and be unlikely to break.Tropoelastin (tropoelastin) is the precursor of solubility elastin, comprise two structural domains of hydrophobicity and wetting ability, become insoluble elastin by lysyl oxidase catalysis when it is positioned extracellular space, the fiber that promptly highly is cross-linked is former.Class elastin polypeptide (elastin-like polypeptides, ELPs) be that the circulation that comes of deriving of the hydrophobic domains of tropoelastin repeats aminoacid sequence, the polypeptide polymer that has the stimulation responses ability by the manual method synthetic, mainly repeat to form (Xaa is called " object amino acid ", can be any amino acid except that proline(Pro)) by Val-Pro-Gly-Xaa-Gly pentapeptide segment unit polyphone.ELPs is the class in numerous stimulation responses polymkeric substance, because it is the recombinant polypeptide by the amino acid coding, can accurately control the composition and the length of ELPs sequence on molecular level, thereby have bigger advantage than other polymkeric substance.The reversible phase transformation can take place along with the rising of temperature in ELPs, promptly becomes insoluble aggregate from solvable state, and this temperature is transformation temperature (Tt).The ELP fusion rotein is called ELPylation again, promptly connects ELPs at the C-of target protein end or N-end, and the ELP fusion rotein has been inherited the characteristic of ELP, promptly also can become insoluble aggregate from solvable state.By changing environmental factorss such as temperature and ionic strength, thereby cause the dissolving of ELP fusion rotein generation reversibility, this process be called the reversible transformation circulation (inverse transition cycling, ITC).By the ITC circular treatment, ELP fusion rotein set physical efficiency is collected by simple centrifugal or ultrafiltration, and can heavily be dissolved in the damping fluid when being lower than transformation temperature or change other envrionment conditionss.Therefore utilize this characteristic of elastin, can satisfy the proteic demand of industrial purification of target simple, quick and in enormous quantities more by taking turns the ITC purge process.Still there is not the report that utilizes class elastin label technology purifying Sumylact L at present both at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing class elastin label purification of Recombinant Sumylact L, further reduce the production cost and the use cost of Sumylact L, realize separation and purification Sumylact L on a large scale.
Above-mentioned purpose of the present invention is achieved by following scheme:
A kind of method of utilizing class elastin label purification of Recombinant Sumylact L may further comprise the steps:
(1) with lactase gene and class elastin label ELP[V
5-80] be connected to successively on the prokaryotic expression carrier pET-32a (+), be built into Sumylact L fusion expression vector pET32a-lactase gene-ELP[V
5-80];
(2) the expression vector electric shock transformed into escherichia coli expression strain that builds, the positive transformant of identifying then carries out self-induction expressed 50-80 hour;
(3) collect thalline, use ultrasonic disruption, centrifugal removal insoluble substance, obtaining the soluble cell lysate is crude enzyme liquid, places 4 ℃ of refrigerators standby;
(4) determine the sedimentary transformation temperature of Sumylact L crude enzyme liquid (Tt);
(5) with ITC circulation purifying Sumylact L recombinant protein.
Lactase gene of the present invention is the lactase gene that derives from various microorganisms, and preferred lactase gene is tt412; This gene has the nucleotide sequence shown in the SEQ ID NO:1, and its amino acid sequence coded is shown in SEQ ID NO:2.
The preferred expression strain of intestinal bacteria of the present invention is BLR (DE3), and this bacterial strain is recA
-The bacterium that derives.
Self-induction expression condition of the present invention is: transformant is inoculated in the triangular flask that contains the TB substratum, 150-300rpm, 30-37 ℃ cultivation is until OD
600Reach 0.5-0.8, under 150rpm, 20-30 ℃ condition, carry out self-induction then and express 50-80h.
Microorganism collection condition of the present invention is: under 4-8 ℃, the centrifugal 5-10min of 6000-10000g collects thalline, thalline with 30mL, 50mM Tris damping fluid (pH8.0) washing once after, it is frozen standby to put into-80 ℃ of refrigerators.
Ultrasonic disruption condition of the present invention is: power 4-8W, broken time 2-4s, broken 4-8s at interval, total time 10-20min.
The mensuration process of transformation temperature of the present invention is: get the crude enzyme liquid of 3 parts of 1ml, add equal-volume respectively but different (the NH of concentration
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be respectively 0.5-1.0M, 1.0-2.0M and 1.5-3.0M, be the OD of gradient recombinant protein aqueous solution when measuring with 2 ℃ from 16 ℃ to 40 ℃
600OD under certain temperature
600Reach at 60 ℃ of recombinant protein aqueous solution OD
600A half, this temperature is transformation temperature Tt.
The present invention obtains the high Sumylact L of purity by the above ITC circulation of four-wheel.
The inventive method is simple, quick, efficient, and the rate of recovery that the total enzyme of Sumylact L is lived can reach 55-65%; The protein purification multiple is 2.0-3.0 times.The present invention not only is applicable to the purifying of reorganization Sumylact L, also can be applicable to the separation and the purifying of other recombinant protein.Therefore, the present invention can be used for separation and purification Sumylact L and other industrial enzyme preparation on a large scale, has higher actual application value in zymin is produced.
Description of drawings
Fig. 1 is the structure flow process of Sumylact L fusion expression vector;
Fig. 2 is the principle schematic of the synthetic ELPs polymkeric substance of RDL method;
Fig. 3 is an ITC purge process synoptic diagram;
Fig. 4 is the SDS-PAGE electrophoretic analysis figure of the Sumylact L after 1-4 wheel ITC purification process;
Wherein, M is high molecular standard protein Marker (a DaLian, China TaKaRa company product), and SL is the solubility supernatant after the fragmentation, i.e. Sumylact L crude enzyme liquid; CS is through ITC circulation final step cold centrifugal (cold spin, CS) the Sumylact L fusion rotein of the purifying after the processing; The digitized representation ITC round-robin number of times of back.
Embodiment
Embodiment one
The preferred lactase gene tt412 of present embodiment, the reorganization Sumylact L that this gene produced have under the low temperature active high, its optimal pH (7.0) near the pH value (6.7-6.8) of crude milk, tolerate the various ion (Na that contained in the crude milk
+, K
+, Ca
2+Deng) etc. series of advantages.
The preferred coli expression carrier pET-32a of prokaryotic expression carrier (+), this carrier has the T7 strong promoter, makes that the expression of exogenous gene amount on the carrier is very high, thereby is beneficial to follow-up purification step.
The preferred expression strain of escherichia coli expression bacterial strain is BLR (DE3), and this bacterial strain is recA
-The bacterium that derives, can improve single plasmid expression level, and can stablize the plasmid that has tumor-necrosis factor glycoproteins or reduce because the losing of the caused DE3 prophage of expression product of plasmid.
Step 1: be built into Sumylact L fusion expression vector pET32a-tt412-ELP[V
5-80].
Lactase gene tt412 shown in the SEQ ID No:1 is building up on the coli expression carrier pET-32a (+) (the Novagen company product in U.S. Madison city), obtains recombinant vectors pET32a-tt412; Again the SFI fragment shown in the SEQ ID NO:3 is connected on the recombinant vectors pET32a-tt412, obtains recombinant vectors pET32a-tt412-SFI; Class elastin label dna sequence dna ELP[V shown in the synthetic SEQ ID NO:4
5-10]; ' end is with an EcoRI restriction enzyme site to this dna sequence dna 5, and 3 ' end is with a HindIII restriction enzyme site.After gene is synthetic, utilize the EcoRI and the HindIII restriction enzyme site at gene two ends to be cloned on the pUC18 carrier (the GenScript company product in U.S. Piscataway city), and employing RDL (recursive directional ligation, cyclic orientation connects, the relevant paper that the details of this method can be delivered with reference to Meyer and Chilkoti2002) method is finished the polymerization of a series of ELPs on the pUC18 carrier, until obtaining class elastin label ELP[V
5-80]; With recombinant vectors pET32a-tt412-SFI and class elastin label ELP[V
5-80] be connected, form Sumylact L fusion expression vector pET32a-tt412-ELP[V
5-80].Above-mentioned vector construction process as shown in Figure 1, the principle of the synthetic ELPs polymkeric substance of RDL method is as shown in Figure 2.
Step 2: the expression vector electric shock transformed into escherichia coli expression strain BLR (DE3) (the Novagen company product in U.S. Madison city) that builds, the transformant of identifying then is inoculated in the 250ml triangular flask that contains 100ml TB substratum, 200rpm, 32 ℃ of cultivations are until OD
600Reach 0.8, under 150rpm, 21 ℃ of conditions, carry out self-induction expression 65h then.
Above-mentioned used TB substratum can be prepared by following process: prepare every liter of substratum, add tryptone 12g, yeast extract 24g, glycerine 4ml in the 900ml deionized water.Shaking container dissolves solute fully.Steam sterilizing 20min under 121 ℃ of temperature then.When being chilled to below 60 ℃ or 60 ℃, solution adds the aseptic 0.17M KH of 100ml
2PO
4With 0.72M K
2HPO
4(compound method of this mixing solutions is mixing solutions: with 90ml deionized water dissolving 2.31g KH
2PO
4With 12.54g K
2HPO
4, after the dissolving, be settled to 100ml and steam sterilizing 20min under 121 ℃ of temperature fully with deionized water).
The step 3:4 ℃ of centrifugal 5min of following 8000g collects thalline, and thalline washs once with 30ml, 50mM Tris damping fluid (pH 8.0), and it is frozen standby to put into-80 ℃ of refrigerators.After the thalline room temperature thawed, be resuspended in 5ml, the 50mM Tris damping fluid (pH 8.0), (condition is cell suspension: 4W with ultrasonic disruption, the broken time of 2s, interval 4s, total time 10min), again with the centrifugal 20min of 16000g to remove insoluble substance such as cell debris, crude enzyme liquid places 4 ℃ of refrigerators standby.
The used damping fluid of above-mentioned washing and resuspended thalline is 50mM Tris damping fluid (pH 8.0), and this damping fluid can be prepared by following process: 0.61g Tris alkali is dissolved in the 50ml deionized water, obtains 50ml, 0.1M Tris alkaline solution.(compound method of 0.1M hydrochloric acid is that to add the 86.2ml mass percent in the 900ml deionized water be 36% concentrated hydrochloric acid, adds deionized water and is settled to 1L, promptly is made into 1M hydrochloric acid to add 29.2ml 0.1M hydrochloric acid in this solution.10 times of 1M hydrochloric acid dilutions are 0.1M hydrochloric acid), behind the mixing, add water volume is transferred to 100ml.
Step 4: determine the sedimentary transformation temperature of Sumylact L crude enzyme liquid.
Get 3 parts of 1ml above-mentioned make crude enzyme liquid, add equal-volume respectively but different (the NH of concentration
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be respectively 0.5M, 1.2M, 2.0M, be the OD of gradient recombinant protein aqueous solution when measuring with 2 ℃ from 16 ℃ to 40 ℃
600OD under certain temperature
600Reach at 60 ℃ of recombinant protein aqueous solution OD
600A half, this temperature is transformation temperature Tt.Determine that by aforesaid method the sedimentary transformation temperature of crude enzyme liquid is 32 ℃.
Step 5: with ITC circulation purifying Sumylact L recombinant protein.
Get the crude enzyme liquid that 1ml step 3 makes, add isopyknic 1M (NH
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be 0.5M.At the definite 32 ℃ of following water-bath 10min of transformation temperature of step 4, the centrifugal 8min of 14000g under room temperature then.Remove supernatant, precipitation is dissolved in ice-cold 30mL, the 50mMTris damping fluid (pH8.0) again, then in 4 ℃ of centrifugal 15min of following 14000g, further to remove insoluble substance.This is one and takes turns the ITC circulation.The gained supernatant continues to carry out purifying with the ITC method.After taking turns ITC circulation through 4, can obtain the very high Sumylact L of purity.The process of ITC method purifying Sumylact L as shown in Figure 3.Through the Sumylact L after the 1-4 wheel ITC circular treatment, its yield and purity situation are as shown in Figure 4.As calculated, the rate of recovery that the total enzyme of Sumylact L is lived in the present embodiment can reach about 58.42%.The protein purification multiple is 2.16 times.Concrete data are as shown in table 1.
In present embodiment and the following examples, the measuring method that the Sumylact L enzyme is lived in the step 5 is: after 100 times of Sumylact L enzyme liquid dilutions to be measured, get 100 μ l, join ONPG (O-Nitrophenyl-β-D-galactopyranoside of 400 μ l 0.25%, ONPG, o-NP-β-D-galactoside) in the solution, places 15min in 46 ℃ of waters bath with thermostatic control, add 500 μ l, 10% Na
2CO
3The solution termination reaction is at OD
405Colorimetric is also calculated enzyme activity.Under these conditions, to produce the required enzyme amount of 1 μ mol o-NP be enzyme unit (U) that lives to Sumylact L per minute hydrolysis ONPG.
Table 1 is through 4 the purifying situations of reorganization Sumylact L after taking turns the ITC circular treatment
Annotate: above all data are the mean value of three measurement results.
Embodiment two:
Present embodiment treatment step and embodiment 1 substantially roughly the same, step 1 is the same with embodiment 1, the difference of other treatment steps also is the difference on used concrete parameter:
(2) the expression vector electric shock transformed into escherichia coli expression strain BLR (DE3) that builds, the transformant of identifying then is inoculated in the 250ml triangular flask (containing 100ml TB substratum), and 180rpm, 30 ℃ of cultivations are until OD
600Reach 0.5, under 150rpm, 25 ℃ of conditions, carry out self-induction expression 75h then.
(3) 6 ℃ of centrifugal 10min of following 6000g collect thalline, thalline washs once with 30ml, 50mM Tris damping fluid (pH8.0), and be resuspended in 5ml, the 50mM Tris damping fluid (pH8.0), cell suspension ultrasonic disruption (8W, the broken time of 3s, interval 5s, total time 15min), the centrifugal 18min of 12000g is with insoluble substances such as removal cell debriss, and crude enzyme liquid places 4 ℃ of refrigerators standby.
(4) get 3 parts of 1ml above-mentioned make crude enzyme liquid, add equal-volume respectively but different (the NH of concentration
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be respectively 0.5M, 1.2M, 2.0M, be the OD of gradient recombinant protein aqueous solution when measuring with 2 ℃ from 16 ℃ to 40 ℃
600OD under certain temperature
600Reach at 60 ℃ of recombinant protein aqueous solution OD
600A half, this temperature is transformation temperature Tt.Determine that by aforesaid method the sedimentary transformation temperature of crude enzyme liquid is 35 ℃.
(5) with ITC method purifying Sumylact L recombinant protein.Get the crude enzyme liquid that 1ml step 3 makes, add an amount of 1M (NH
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be 0.8M.At 35 ℃ of following water-bath 12min of transformation temperature, the centrifugal 10min of 12000g under room temperature then.Remove supernatant, precipitation is dissolved in ice-cold 30ml, the 50mM Tris damping fluid (pH8.0) again, then in 4 ℃ of centrifugal 20min of following 12000g, further to remove insoluble substance.This is one and takes turns the ITC circulation.The gained supernatant continues to carry out purifying with the ITC method.After taking turns ITC circulation through 4, can obtain the very high Sumylact L of purity.As calculated, as calculated, the rate of recovery that the total enzyme of Sumylact L is lived in the present embodiment can reach about 61.66%.The protein purification multiple is 2.38 times.Concrete data are as shown in table 2.
Table 2 is through 4 the purifying situations of reorganization Sumylact L after taking turns the ITC circular treatment
Annotate: above all data are the mean value of three measurement results.
Embodiment three:
Present embodiment treatment step and embodiment 1 substantially roughly the same, step 1 is the same with embodiment 1, the difference of other treatment steps also is the difference on used concrete parameter:
(2) the expression vector electric shock transformed into escherichia coli expression strain BLR (DE3) that builds, the transformant of identifying then is inoculated in the 250ml triangular flask (containing 100ml TB substratum), and 250rpm, 37 ℃ of cultivations are until OD
600Reach 0.6, under 150rpm, 30 ℃ of conditions, carry out self-induction expression 65h then.
(3) 8 ℃ of centrifugal 10min of following 10000g collect thalline, thalline washs once with 30ml, 50mM Tris damping fluid (pH8.0), and be resuspended in 5ml, the 50mM Tris damping fluid (pH8.0), cell suspension ultrasonic disruption (power 6W, the broken time of 4s, interval 8s, total time 20min), the centrifugal 25min of 14000g is with insoluble substances such as removal cell debriss, and crude enzyme liquid places 4 ℃ of refrigerators standby.
(4) get 3 parts of 1ml above-mentioned make crude enzyme liquid, add equal-volume respectively but different (the NH of concentration
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be respectively 0.5M, 1.2M, 2.0M, be the OD of gradient recombinant protein aqueous solution when measuring with 2 ℃ from 16 ℃ to 40 ℃
600OD under certain temperature
600Reach at 60 ℃ of recombinant protein aqueous solution OD
600A half, this temperature is transformation temperature Tt.Determine that by aforesaid method the sedimentary transformation temperature of crude enzyme liquid is 37 ℃.
(5) with ITC method purifying Sumylact L recombinant protein.Get the crude enzyme liquid that 1ml step 3 makes, add an amount of 2M (NH
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be 1.2M.At 37 ℃ of following water-bath 15min of transformation temperature,, the centrifugal 5min of 16000g under room temperature then.Remove supernatant, precipitation is dissolved in ice-cold 30ml, the 50mM Tris damping fluid (pH8.0) again, then in 4 ℃ of centrifugal 10min of following 16000g, further to remove insoluble substance.This is one and takes turns the ITC circulation.The gained supernatant continues to carry out purifying with the ITC method.After taking turns ITC circulation through 5, can obtain the Sumylact L behind the purifying.As calculated, the rate of recovery that the total enzyme of Sumylact L is lived in the present embodiment can reach about 63.79%.The protein purification multiple is 2.53 times.Concrete data are as shown in table 3.
Table 3 is through 4 the purifying situations of reorganization Sumylact L after taking turns the ITC circular treatment
Annotate: above all data are the mean value of three measurement results.
Embodiments of the present invention are not limited thereto, and according to foregoing of the present invention, according to the ordinary skill knowledge and the customary means of this area, are not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, and the present invention can also have other embodiment; Can be as lactase gene for deriving from the lactase gene of various microorganisms, as derive from the beta-galactosidase gene LAC4 of Kluyveromyces lactis (Kluyveromyces lactis), derive from the beta-galactosidase gene lacL of Lactobacillus reuteri, derive from beta-galactosidase gene 32c β-gal of AntarcticArthrobacter sp.32c etc.Coli expression carrier can be other protokaryon efficient expression vector, as the ChampionTM pET Expression System series prokaryotic expression carrier of Invitrogen company (U.S. Carlsbad city), the pEGX series prokaryotic expression carrier of GE company (pittsburgh,U.S.A city) etc.The escherichia coli expression bacterial strain can adopt other derivative strain, as the BL21 (DE3) of U.S. Novagen company and Rosetta (DE3) etc.Therefore, the present invention can also make modification, replacement or the change of other various ways, all drops within the rights protection scope of the present invention.
Claims (8)
1. method of utilizing class elastin label purification of Recombinant Sumylact L is characterized in that may further comprise the steps:
(1) with lactase gene and class elastin label ELP[V
5-80] be connected to successively on the prokaryotic expression carrier pET-32a (+), be built into Sumylact L fusion expression vector pET32a-lactase gene-ELP[V
5-80];
(2) the expression vector electric shock transformed into escherichia coli expression strain that builds, the positive transformant of identifying then carries out self-induction expressed 50-80 hour;
(3) collect thalline, use ultrasonic disruption, centrifugal removal insoluble substance, obtaining the soluble cell lysate is crude enzyme liquid, places 4 ℃ of refrigerators standby;
(4) determine the sedimentary transformation temperature of Sumylact L crude enzyme liquid (Tt);
(5) with ITC circulation means purifying Sumylact L recombinant protein.
2. the method for purification of Recombinant Sumylact L according to claim 1 is characterized in that: described lactase gene is the lactase gene that derives from various microorganisms, and preferred lactase gene is tt412; This gene has the nucleotide sequence shown in the SEQ ID NO:1, and its amino acid sequence coded is shown in SEQ ID NO:2.
3. the method for purification of Recombinant Sumylact L according to claim 1 is characterized in that: the preferred expression strain of described intestinal bacteria is BLR (DE3), and this bacterial strain is the bacterium that derives of recA-.
4. the method for purification of Recombinant Sumylact L according to claim 1 is characterized in that, described self-induction expression condition is: transformant is inoculated in the triangular flask that contains the TB substratum, 150-300rpm, 30-37 ℃ cultivation is until OD
600Reach 0.5-0.8, under 150rpm, 20-30 ℃ condition, carry out self-induction then and express 50-80h.
5. the method for purification of Recombinant Sumylact L according to claim 1, it is characterized in that, described microorganism collection condition is: under 4-8 ℃, the centrifugal 5-10min of 6000-10000g collects thalline, after thalline washed once with 30ml, 50mM Tris damping fluid (pH8.0), it was frozen standby to put into-80 ℃ of refrigerators.
6. the method for purification of Recombinant Sumylact L according to claim 1 is characterized in that, described ultrasonic disruption condition is: power 4-8W, broken time 2-4s, broken 4-8s at interval, total time 10-20min.
7. the method for purification of Recombinant Sumylact L according to claim 1 is characterized in that, the mensuration process of described transformation temperature is: get the crude enzyme liquid of 3 parts of 1ml, add equal-volume respectively but different (the NH of concentration
4)
2SO
4Solution makes (NH in the crude enzyme liquid
4)
2SO
4Final concentration be respectively 0.5-1.0M, 1.0-2.0M and 1.5-3.0M, be the OD of gradient recombinant protein aqueous solution when measuring with 2 ℃ from 16 ℃ to 40 ℃
600OD under certain temperature
600Reach at 60 ℃ of recombinant protein aqueous solution OD
600A half, this temperature is transformation temperature Tt.
8. the method for purification of Recombinant Sumylact L according to claim 1 is characterized in that, by the circulation of the ITC more than the four-wheel, obtains the high Sumylact L of purity.
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