CN101418290A - High efficiency ELP fusion protease as well as preparation and application thereof - Google Patents
High efficiency ELP fusion protease as well as preparation and application thereof Download PDFInfo
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Abstract
The invention relates to an efficient fusion protease, namely an ELP-3C protease which is obtained through the fusion expression between elastin-like polypeptide (ELP) and human rhinovirus 3C protease. As the ELP is adopted as a purification tag, the fusion protease can be purified without the assistance of a chromatography separation technology, thereby reducing the cost of purification. The ELP-3C protease can be applied to cutting fusion protein; and after enzyme cutting of substrate protein, the ELP-3C protease is triggered to precipitate through simply increasing the concentration of salt ions and is removed through centrifuging, wherein the process is performed without the assistance of the chromatographic separation technology, and the ELP-3C protease can be used for the enzyme cutting continuously for multiple times and keeps the enzyme activity after multiple times of the enzyme cutting, thus the cost of large-scale protein purification can be reduced.
Description
Technical field
The present invention relates to a kind of fusion protease of reorganization, and the application of this proteolytic enzyme in cleavage of fusion proteins.
Background technology
Along with large scale sequencing technical development and widespread use, the Human Genome Project and other species comprise that genome plans such as sea urchin, zebra fish, lancelet also finish in succession.Direct result to the gene order-checking of these species is to produce thousands of genes, to illustrating of these gene functions, helps us to understand the position of species in evolutionary process; Research to the gene that causes disease helps to understand pathogeny, seeks the target spot of treatment, exploitation treatment corresponding treatment medicine; Simultaneously, a large amount of pharmaceutical protein genes remains also that we remove to study its physiologically active, and action target spot, for the medicine of development of new provides the basis.The method of gene functional research is a lot, comprises the gene knockout technology, the research of protein science, RNA interference etc.One of them the most direct research method be the gene of being studied is carried out recombinant expressed, thereby obtain to carry out the research of physiology and biochemical activity and its function is illustrated in its analysis of carrying out structure biology after the corresponding proteins.Because the otherness of species gene, not all gene can both successfully be expressed.Gene is not expressed, the low or insoluble inclusion body of the unstable formation of expressed proteins of expressing quantity, and these phenomenons often occur in heterologous expression system, express eukaryotic gene especially in prokaryotic expression system.Development along with the DNA recombinant technology, goal gene and the gene of expressing affinity tag can be merged, be cloned in then and carry out amalgamation and expression in the expression vector, the target protein that these affinity tag not only can help to be merged folds, improve the solubility and the expression amount of target protein, simultaneously affinity tag can with the resin-bonded of corresponding affinity chromatography, play the effect of single step purification fusion rotein.Affinity purification label commonly used comprise glutathione-S-transferase (Glutathione-S-Transferase, GST), maltose binding protein (Maltose-binding Protein, MBP), His label etc.Utilize proteolytic enzyme that the fusion rotein that contains corresponding protein enzyme restriction enzyme site is cut at last, target protein and affinity tag albumen sepn are opened, remove affinity tag and proteolytic enzyme obtains target protein by further chromatographic separation.
Affinity chromatography is a kind of very effective separation method, and the situation at the physico-chemical property that does not need to understand institute's fusion rotein just can realize the single step purification fusion rotein, and this purification technique is widely used.But using the affinity chromatography isolation technique needs expensive affine resin and corresponding purifier apparatus, has limited like this to express simultaneously that multiple protein is used for high-throughout structure biology research and high-throughout pharmaceutical protein screens.1999, (Elastin Like Polypeptides, ELP), this label originated from the multiple pentapeptide " Val-Pro-Gly-Val-Gly " of elastin to have reported a heat sensitive purification tag elastin-like polypeptide.ELP can experience a reversible transformation process, is called anti-phase transition temperature (Inverse TemperatureTransition).When temperature is lower than transformation temperature (Transition temperature, Tt) time, ELP shows highly solvable in liquid phase, yet when temperature is higher than Tt, hydrophilic ELP then can dewater and aggegation takes place, and the ELP fusion rotein also has this specific character, but it is to be noted when undergoing phase transition to be that ELP condenses and sex change or precipitation do not take place for the foreign protein that merged.Utilize this specific character of ELP, can make things convenient for and utilize the ELP label to come purifying ELP fusion rotein apace, and do not need chromatographic separation in the purge process and avoided expensive affine resin and purifier apparatus, simultaneously the ELP fusion rotein concentrate and exchange buffering liquid also becomes very simple.Trigger the aggegation of ELP fusion rotein by heating or raising salt ionic concentration, separate in expressive host albumen by the centrifugal ELP of making fusion rotein, with the cryogenic solution of less salt sedimentary ELP fusion rotein is dissolved again, carry out again centrifugally removing insoluble albumen and obtaining the ELP fusion rotein, can obtain purer ELP fusion rotein by repeated trigger precipitation, centrifugal and heavy molten step, this purge process be referred to as anti-phase circulation (Inverse TransitionCycling, ITC).
In order to eliminate physiologically active, physical structure and the immunogenic influence of purification tag to target protein, purified fusion rotein need be removed purification tag.Cutting fusion tag method comprises the method for chemical process and zymetology.The condition of chemical process cutting is more violent, cause easily protein denaturation and albumen side chain chemically modified and cut; Qie Ge site also only is defined in one or two specific amino acid in addition, causes easily target protein inside is cut.So being only limited to, this method separates little peptide and the little albumen of molecular weight.On the contrary, the condition of proteolytic enzyme cutting is just gentle a lot of with respect to chemical method, and the enzyme of proteolytic enzyme cuts special height, has the multiple protein enzyme available, is first-selected so use proteolytic enzyme in the fusion rotein cutting, although business-like proteolytic enzyme is relatively more expensive.ERC group virus 3C proteolytic enzyme (Human Rhinovirus 14 3C Protease) is a kind of proteolytic enzyme efficiently.3C proteolytic enzyme derives from ERC group virus, is a kind of L-Cysteine HCL Anhydrous, and the shear protein sequence of its identification is Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro, and cleavage site is between Gln-Gly.Business-like 3C proteolytic enzyme merges (GST-3C) with GST, is referred to as PreScission Protease, contains the PreScission restriction enzyme site in the pGEX series fusion expression vector commonly used.3C proteolytic enzyme still keeps very high enzyme to cut activity, even effects of ion concentration up to 0.5M, also keeps enzyme to cut activity under low temperature and high salt condition.Compare other proteolytic enzyme, the 3C enzyme has advantages such as identification specificity is stronger, cutting efficiency is higher, reaction conditions gentleness.
Summary of the invention
The object of the present invention is to provide a kind of ELP-3C of fusion protease efficiently proteolytic enzyme.
Another object of the present invention is to provide the preparation method of this fusion protease.
The 3rd purpose of the present invention is to provide the application of this fusion protease in cleavage of fusion proteins.
According to an aspect of the present invention, merge the ELP-3C proteolytic enzyme of encoding by elastin-like polypeptide (ELP) gene and ERC group virus 3C proteinase gene, its aminoacid sequence is shown in SEQ ID NO.3.
According to another aspect of the present invention, the preparation method of described ELP-3C proteolytic enzyme may further comprise the steps:
(1) with the gene clone of ELP-3C protease-encoding to prokaryotic expression carrier pET23a, the construction expression plasmid;
(2) described expression plasmid is transformed into e. coli strains BL21 (DE3), and carries out culture expression;
(3) described intestinal bacteria are carried out ultrasonic degradation, cracking bacterium liquid carries out low-temperature centrifugation, gets supernatant liquor;
(4) trigger the aggegation of ELP-3C proteolytic enzyme by the salt ionic concentration that improves described supernatant liquor, room temperature is centrifugal, removes supernatant liquor, and throw out dissolves again with low temperature less salt solution;
(5) repeat described aggegation, centrifugal and again the dissolved step until obtaining pure ELP-3C proteolytic enzyme.
Among the present invention, the culture expression of the e. coli bl21 after the conversion (DE3) be with single colony inoculation in the LB of Amp+ liquid enriched medium, 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria; Get kind of daughter bacteria and be inoculated in the fresh Amp+TB substratum, 37 ℃ of thermal agitation amplification culture to O.D.600 be 1.0 o'clock, temperature is transferred to 18 ℃ cultivated 24 hours.
Gene clone technology of the present invention: the clone implements the experiment condition described in the chamber handbook (Sambrook, et al.1989, Molecular Cloing.Cold Spring Harbor Labroratory Press.USA) with reference to the Sambrook equimolecular.
Expression plasmid clone method of the present invention:, use CaCl with reference to Sambrook (Sambrook, et al.1989, Molecular Cloing.Cold Spring Harbor Labroratory Press.USA) method
2Method with plasmid Transformed E .coli.DH50 or BL21 (DE3) bacterial strain, transform bacterial strain with the LB culture medium culturing that contains penbritin (100 μ g/mL).
According to a third aspect of the present invention, provide the application of ELP-3C proteolytic enzyme in cleavage of fusion proteins.
The ELP-3C proteolytic enzyme that the present invention obtained is behind cleavage of fusion proteins, by improving the triggering ELP-3C proteolytic enzyme aggegation of solution salt ionic concn and being removed by centrifugal.
The ELP-3C proteolytic enzyme that the present invention obtained can carry out the several times enzyme continuously to be cut, and keeps enzyme to cut activity.
The present invention gropes the expression condition of ELP-3C fusion protease and optimizes, and by optimizing conditions such as incubation time, induction time and temperature, make the expression amount of ELP-3C proteolytic enzyme higher, and the overwhelming majority is in solvable state.The purifying of preparation-obtained ELP-3C proteolytic enzyme need not to use the filler and the corresponding apparatus of chromatographic separation, thereby can reduce the purifying cost of ELP-3C proteolytic enzyme.
The cleavage of fusion proteins experiment confirm: the optimal concentration of ELP-3C proteolytic enzyme cutting fusion rotein is an ELP-3C proteolytic enzyme: fusion rotein=1: 100 (w/w), and ELP-3C proteolytic enzyme can efficiently cut fusion roteins such as GST-ELP, ELP-GST, ELP-MBP and ELP-TRX under 1: 100 (w/w) concentration; Can trigger ELP-3C and ELP aggegation and be removed by improving the solution salt ionic concn, thereby realize need not removing under the chromatographic separation technology ELP-3C proteolytic enzyme by centrifugal.In addition, enzyme is cut the fusion rotein experiment confirm continuously: reclaim behind the ELP-3C proteolytic enzyme cutting ELP-GFP albumen, the ELP-3C proteolytic enzyme after the recovery still has enzyme and cuts activity, and can carry out 6 enzymes continuously and cut.Therefore, ELP-3C proteolytic enzyme can be used as the instrument that purifying does not have the label target protein, and can reduce the cost of large-scale protein purifying.
Description of drawings
Fig. 1 is the abduction delivering and the purifying of ELP-3C proteolytic enzyme and is applied to the proteic SDS-PAGE electrophorogram of purifying GST.M: protein molecular weight standard; Total mycoprotein of 1:ELP-3C proteolytic enzyme abduction delivering; The cracking supernatant of 2:ELP-3C proteolytic enzyme abduction delivering; 3: with the centrifugal supernatant behind the salt deposit E LP-3C proteolytic enzyme; 4: the ELP-3C proteolytic enzyme behind the purifying; 5: the GST-ELP behind the purifying; 6:ELP-3C proteolytic enzyme enzyme is cut GST-ELP; 7: the precipitation enzyme is cut product; 8: the GST of purifying.
Fig. 2 cuts the SDS-PAGE electrophorogram that concentration is groped for the best enzyme that ELP-3C proteolytic enzyme enzyme is cut the GST-ELP fusion rotein.1:GST-ELP; 2:ELP-3C proteolytic enzyme: GST-ELP=1: 10 (w/w); 3:ELP-3C proteolytic enzyme: GST-ELP=1: 50 (w/w); 4:ELP-3C proteolytic enzyme: GST-ELP=1: 100 (w/w); 5:ELP-3C proteolytic enzyme: GST-ELP=1: 500 (w/w); 6:ELP-3C proteolytic enzyme: GST-ELP=1: 1000 (w/w); 7:ELP-3C proteolytic enzyme: GST-ELP=1: 5000 (w/w); 8:ELP-3C proteolytic enzyme: GST-ELP=1: 10000 (w/w).
Fig. 3 is for utilizing the proteic SDS-PAGE electrophorogram of ELP-3C protease purification GFP.M: protein molecular weight standard; Total mycoprotein of 1:ELP-GFP abduction delivering; The cracking supernatant of 2:ELP-GFP abduction delivering; 3: with the centrifugal supernatant behind the salt deposit E LP-GFP; 4: the ELP-GFP behind the purifying; 5:ELP-3C proteolytic enzyme enzyme is cut ELP-GFP; 6: the precipitation enzyme is cut product; 7: the GFP of purifying.
The GFP albumen that Fig. 4 detects down for fluorescent microscope.
Fig. 5 is for utilizing the proteic SDS-PAGE electrophorogram of ELP-3C protease purification MBP.M: protein molecular weight standard; Total mycoprotein of 1:ELP-MBP abduction delivering; The cracking supernatant of 2:ELP-MBP abduction delivering; 3: with the centrifugal supernatant behind the salt deposit E LP-MBP; 4: the ELP-MBP behind the purifying; 5:ELP-3C proteolytic enzyme enzyme is cut ELP-MBP; 6: the precipitation enzyme is cut product; 7: the MBP of purifying.
Fig. 6 is for utilizing the proteic SDS-PAGE electrophorogram of ELP-3C protease purification TRX.M: protein molecular weight standard; Total mycoprotein of 1:ELP-TRX abduction delivering; The cracking supernatant of 2:ELP-TRX abduction delivering; 3: with the centrifugal supernatant behind the salt deposit E LP-TRX; 4: the ELP-TRX behind the purifying; 5:ELP-3C proteolytic enzyme enzyme is cut ELP-TRX; 6: the precipitation enzyme is cut product; 7: the TRX of purifying.
Fig. 7 cuts the proteic SDS-PAGE electrophorogram of GFP of purifying for continuous enzyme.1: for the first time ELP-3C proteolytic enzyme enzyme is cut the GFP of purifying behind the ELP-GFP; 2: for the second time ELP-3C proteolytic enzyme enzyme is cut the GFP of purifying behind the ELP-GFP; 3: ELP-3C proteolytic enzyme enzyme is cut the GFP of purifying behind the ELP-GFP for the third time; 4: the four times ELP-3C proteolytic enzyme enzymes are cut the GFP of purifying behind the ELP-GFP; 5: the five times ELP-3C proteolytic enzyme enzymes are cut the GFP of purifying behind the ELP-GFP; 6: the six times ELP-3C proteolytic enzyme enzymes are cut the GFP of purifying behind the ELP-GFP.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions among the following embodiment, usually according to normal condition, for example Sambrook equimolecular clone implements the condition described in the handbook of chamber.
The structure and the alternate host bacterium of embodiment 1:ELP-3C proteolytic enzyme expression vector
According to the synthetic ELP gene (Meyer of people's reported method such as Meyer in 1999 and Chilkoti, D.E., and Chilkoti, A.1999.Purification of recombinant proteins by fusion with thermally-responsive polypeptides.NatBiotechnol 17:1112-1115.), its nucleotide sequence is shown in SEQ ID NO.1.After the EcorI by ELP gene N end and the HindIII enzyme of C end are cut, clone the corresponding restriction enzyme site in commercialization prokaryotic expression carrier pET23a and form the pET23a-ELP carrier.The pET23a-ELP plasmid is transformed into e.colistraindh5, after culture expression, extracts the pET23a-ELP plasmid.Simultaneously, gene order (accession number: M12168) synthetic 3C proteinase gene according to the ERC group virus 3C proteolytic enzyme of announcing on the genbank in NCBI, its nucleotide sequence and is cloned in the pbluscript carrier and is formed the pbluscript-3C carrier shown in SEQ ID NO.2.The pbluscript-3C plasmid is transformed into e.colistraindh5, after culture expression, extracts the pbluscript-3C plasmid.The HindIII of application 3C proteinase gene N end and two kinds of restriction endonucleases of NotI of C end carry out enzyme to pbluscript-3C and cut, purifying is obtained the 3C gene fragment clone in the corresponding restriction enzyme site of pET23a-ELP carrier, make the 3C proteinase gene be positioned at the C end of ELP gene, and obtain ELP-3C proteolytic enzyme expression vector pET23a-ELP-3C.
Expression vector pET23a-ELP-3C with making up with reference to Sambrook (Sambrook, et al.1989, Molecularcloing.Cold Spring Harbor Labroratory Press.USA) method, uses CaCl
2Method plasmid is transformed into E.coli.BL21 (DE3) bacterial strain, transform bacterial strain with the LB culture medium culturing that contains penbritin (100 2g/mL).
Embodiment 2: the expression and purification of reorganization ELP-3C proteolytic enzyme
The single colony inoculation of expressive host bacterium BL21 (DE3) that will contain recombinant plasmid pET23a-ELP-3C is in the LB of Amp+ liquid enriched medium, and 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria.Get kind of daughter bacteria and be inoculated in the fresh Amp+TB substratum by 1: 100 volume ratio, 37 ℃ of thermal agitation amplification culture are about at 1.0 o'clock to O.D.600, temperature is transferred to 18 ℃ cultivated 24 hours.Bacterium liquid after 100ml induced carries out centrifugal receipts bacterium, 5000rpm5 minute.With the resuspended thalline of the ice-cold PBS of 8ml, carry out the ultrasonic degradation thalline then, power is 200W, ultrasonic 15 minutes.Cracking bacterium liquid centrifugal 10 minutes of 4 ℃ of 12000g, is removed insoluble tropina.Solid NaCl is added in the centrifuged supernatant, and make it dissolving, strength of solution is 2M, and the cracking supernatant liquor becomes muddiness by clarification at ambient temperature.The cracking supernatant liquor room temperature high speed centrifugation that change is muddy was removed supernatant liquor after 5 minutes.The resuspended precipitation of PBS that adds precooling will precipitate piping and druming and loose, and place on ice till all precipitations are all dissolved.This protein solution centrifugal 5 minutes of 4 ℃ of 12000g, is removed insoluble precipitation.The supernatant liquor of centrifugal gained is repeated to make the albumen aggegation with salt, centrifugal, soluble protein precipitation, the insoluble sedimentary step of centrifugal removal, (see figure 1) till removing all foreign proteins to obtain purer target protein.
Embodiment 3: utilize ELP-3C protease purification GST albumen
The single colony inoculation of pGEX-ELP expressive host bacterium BL21 (DE3) that will contain recombinant plasmid is in the LB of Amp+ liquid enriched medium, and 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria.Get kind of daughter bacteria and be inoculated in the fresh Amp+LB enriched medium by 1: 100 volume ratio, 37 ℃ of thermal agitation amplification culture are about at 1.0 o'clock to O.D.600, add 0.5mMIPTG, 37 ℃ of abduction deliverings 4 hours.Carry out cracking after receiving bacterium, making strength of solution by adding solid NaCl is 2M, triggers GST-ELP aggegation and centrifugal its precipitation that makes, and uses the 3C enzyme cutting buffering liquid to dissolve.Added ELP-3C proteolytic enzyme by 1: 100,5 ℃ of enzymes were cut 16 hours.Enzyme is cut the back and is added solid NaCl to make strength of solution be 2M, triggers ELP and the aggegation of ELP-3C proteolytic enzyme and centrifugal removing.Collecting supernatant liquor is the GST albumen (see figure 1) of purifying.
Embodiment 4:ELP-3C proteolytic enzyme enzyme is cut the suitableeest enzyme of GST-ELP fusion rotein and is cut concentration
Enzyme is cut 100 μ gGST-ELP protein substrates, presses ELP-3C: GST-ELP mass ratio 1: 10, and 1: 50,1: 100,1:: add ELP-3C proteolytic enzyme at 500,1: 1000,1: 5000,1: 10000, cut 16 hours in 5 ℃ of enzymes behind the mixing.Endonuclease reaction carries out SDS-PAGE electrophoresis detection (see figure 2) after finishing.
Embodiment 5: utilize ELP-3C protease purification GFP albumen
The single colony inoculation of expressive host bacterium BL21 (DE3) that will contain recombinant plasmid pET23a-ELP-GFP is in the LB of Amp+ liquid enriched medium, and 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria.Get kind of daughter bacteria and be inoculated in the fresh Amp+TB substratum by 1: 100 volume ratio, 37 ℃ of thermal agitation amplification culture are about at 1.0 o'clock to O.D.600, temperature is transferred to 18 ℃ cultivated 24 hours.Carry out cracking after receiving bacterium, making strength of solution by adding solid NaCl is 2M, triggers ELP-GFP aggegation and centrifugal its precipitation that makes, and uses the 3C enzyme cutting buffering liquid to dissolve.Added ELP-3C proteolytic enzyme by 1: 100,5 ℃ of enzymes were cut 16 hours.Enzyme is cut the back and is added solid NaCl to make strength of solution be 2M, triggers ELP and the aggegation of ELP-3C proteolytic enzyme and centrifugal removing.Collecting supernatant liquor is the GFP albumen (see figure 3) of purifying.The GFP albumen of purifying is put observation under the fluorescent microscope, can detect tangible fluorescence (see figure 4).
Embodiment 6: utilize ELP-3C protease purification MBP albumen
The single colony inoculation of expressive host bacterium BL21 (DE3) that will contain recombinant plasmid pET23a-ELP-MBP is in the LB of Amp+ liquid enriched medium, and 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria.Get kind of daughter bacteria and be inoculated in the fresh Amp+TB substratum by 1: 100 volume ratio, 37 ℃ of thermal agitation amplification culture are about at 1.0 o'clock to O.D.600, temperature is transferred to 18 ℃ cultivated 24 hours.Carry out cracking after receiving bacterium, making strength of solution by adding solid NaCl is 2M, triggers ELP-MBP aggegation and centrifugal its precipitation that makes, and uses the 3C enzyme cutting buffering liquid to dissolve.Added ELP-3C proteolytic enzyme by 1: 100,5 ℃ of enzymes were cut 16 hours.Enzyme is cut the back and is added solid NaCl to make strength of solution be 2M, triggers ELP and the aggegation of ELP-3C proteolytic enzyme and centrifugal removing.Collecting supernatant liquor is the MBP albumen (see figure 5) of purifying.
Embodiment 7: utilize ELP-3C protease purification TRX albumen
The single colony inoculation of expressive host bacterium BL21 (DE3) that will contain recombinant plasmid pET23a-ELP-TRX is in the LB of Amp+ liquid enriched medium, and 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria.Get kind of daughter bacteria and be inoculated in the fresh Amp+TB substratum by 1: 100 volume ratio, 37 ℃ of thermal agitation amplification culture are about at 1.0 o'clock to O.D.600, temperature is transferred to 18 ℃ cultivated 24 hours.Carry out cracking after receiving bacterium, making strength of solution by adding solid NaCl is 2M, triggers making GST-GFP aggegation and centrifugal its precipitation that makes, and uses the 3C enzyme cutting buffering liquid to dissolve.Added ELP-3C proteolytic enzyme by 1: 100,5 ℃ of enzymes were cut 16 hours.Enzyme is cut the back and is added solid NaCl to make strength of solution be 2M, triggers ELP and the aggegation of ELP-3C proteolytic enzyme and centrifugal removing.Collecting supernatant liquor is the TRX albumen (see figure 6) of purifying.
Embodiment 8:ELP-3C proteolytic enzyme reuses active detection
Enzyme is cut 1mg ELP-GFP albumen, presses ELP-3C: the ELP-GFP mass ratio adds ELP-3C proteolytic enzyme at 1: 100.After 20 ℃ of enzymes were cut 4 hours, it was 2M that adding solid NaCl makes enzyme cut strength of solution, and room temperature high speed centrifugation 5 minutes keeps the supernatant sample.Precipitation is resuspended with the ELP-GFP protein solution that contains 1mg, and constantly pressure-vaccum makes till the resolution of precipitate, proceeds 20 ℃ of enzymes behind the adding DTT (final concentration 1mM) and cuts 4 hours.So repeat top step 6 time.The GFP albumen of each purifying is carried out SDS-PAGE protein electrophoresis detection (see figure 7).
Sequence table
＜110〉Zhongshan University
＜120〉a kind of fusion protease of ELP efficiently and preparation and application
<160>3
<210>1
<211>1374
<212>DNA
＜213〉artificial sequence
<220>
<221>
<222>(1)..(1374)
＜223〉ELP gene
<400>1
<210>2
<211>549
<212>DNA
＜213〉ERC group virus (Human Rhinovirus)
<400>2
<210>3
<211>648
<212>PRT
＜213〉artificial sequence
<220>
<221>
<222>(1)..(648)
＜223〉ELP-3C proteolytic enzyme
<400>3
Claims (6)
1, merged the ELP-3C proteolytic enzyme of coding by elastin-like polypeptide (ELP) gene and ERC group virus 3C proteinase gene, its aminoacid sequence is shown in SEQ ID NO.3.
2, the preparation method of the described ELP-3C proteolytic enzyme of claim 1 may further comprise the steps:
(1) with the gene clone of ELP-3C protease-encoding to prokaryotic expression carrier pET23a, the construction expression plasmid;
(2) described expression plasmid is transformed into e. coli strains BL21 (DE3), and carries out culture expression;
(3) described intestinal bacteria are carried out ultrasonic degradation, cracking bacterium liquid carries out low-temperature centrifugation, gets supernatant liquor:
(4) trigger the aggegation of ELP-3C proteolytic enzyme by the salt ionic concentration that improves described supernatant liquor, room temperature is centrifugal, removes supernatant liquor, and throw out dissolves again with low temperature less salt solution;
(5) repeat described aggegation, centrifugal and again the dissolved step until obtaining pure ELP-3C proteolytic enzyme.
3, preparation method according to claim 2 is characterized in that: the described culture expression of step (2) be with single colony inoculation in the LB of Amp+ liquid enriched medium, 37 ℃ of 225rpm cultivated 12 hours, as kind of a daughter bacteria; Get kind of daughter bacteria and be inoculated in the fresh Amp+TB substratum, 37 ℃ of thermal agitation amplification culture to O.D.600 be 1.0 o'clock, temperature is transferred to 18 ℃ cultivated 24 hours.
4, the application of the described ELP-3C proteolytic enzyme of claim 1 in cleavage of fusion proteins.
5, application according to claim 4 is characterized in that: ELP-3C proteolytic enzyme is behind cleavage of fusion proteins, by improving the triggering ELP-3C proteolytic enzyme aggegation of solution salt ionic concn and being removed by centrifugal.
6, according to claim 4 or 5 described application, it is characterized in that: ELP-3C proteolytic enzyme carries out the several times enzyme continuously and cuts.
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| WO2010063172A1 (en) * | 2008-12-02 | 2010-06-10 | 中山大学 | An elp fusion protein and uses thereof |
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| US20080032931A1 (en) * | 1999-08-25 | 2008-02-07 | Steward Lance E | Activatable clostridial toxins |
| US6852834B2 (en) * | 2000-03-20 | 2005-02-08 | Ashutosh Chilkoti | Fusion peptides isolatable by phase transition |
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