CN101942024B - Heparanase II fusion protein and coding gene and expression method thereof - Google Patents
Heparanase II fusion protein and coding gene and expression method thereof Download PDFInfo
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Abstract
The invention discloses a heparanase II fusion protein, and a coding gene and an expression method thereof. The fusion protein (named MBP-HepB) is a protein of a) or b), wherein a) is a protein consisting of an amino acid sequences shown from the 1st position to 1,118th position of a sequence 2 in a sequence table; and b) is a protein in which one or more amino acid is substituted, and/or deleted and/or added in the amino acid sequence of a sequence 2 in a sequence table and which has heparanase activity and derives from the a). The coding gene of the protein is also in the protection scope of the invention. The gene is introduced into the protein heparanase expressed in the recombinant escherichia coli, the enzyme activity reaches 118.4IU/L fermentation liquor, the expression quantity reaches 179mg/L fermentation liquor, and the specific enzyme activity reaches 0.66IU/mg. The one-step purification of the fusion protein can also be realized by affinity separation.
Description
Technical field
The present invention relates to a kind of Heparinase I I fusion rotein and encoding sox and expression method in genetically engineered and the field of fermentation engineering.
Background technology
Heparinase (heparinase) is one type of polysaceharide lyase that acts on heparin (heparin) or Suleparoid (heparan sulfate); In many kinds of mikrobes, find; Comprise excellent bacillus Corynebacterium sp. (high Ningguo etc.; Heparinase produces screening and the fermentation condition of bacterium; Mikrobe journal 1999 Vol.39:64-67), Sphingobacterium sp. Sphingobacterium sp. (high Ningguo etc.; The generation of Sphingobacterium sp. heparinase; Mikrobe journal 2003 Vol.43:813-816), subtilis Bacillus subtilis (Wang Zhongyan; Heparinase produces the screening of bacterium and the research of thick enzymatic property thereof; Sichuan University's journal (natural science version) 2002 Vol.39:777-779), Bacillus circulans Bacillus circulans (Yasutaka Tahara et al.; Purification and characterization of heparinase that degrades both heparin and heparin sulfate from Bacillus circulans BioSci.Biotechnol.Biochem.2002 Vol.66:1181-1184), Bacteroides heparinolyticus Prevotella heparinolytica (Kazuyuki Sugahara et al.; Characterization of heparinase from an oral bacterium Prevotella heparinolytica J.Biochem.1998 Vol.123:283-288), Bacteroides stercoris Bacteroides stercoris HJ-15 (Dong Hyuu Kim et al.; Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15 J.Biochem.2000 Vol.128:323-328) and heparin Flavobacterium Flavobacterium heparinum (Sasiekharan, Ph.D.Thesis R.1991, Havards University).But the heparinase from the heparin Flavobacterium is business-like unique source.
The research of heparinase has crucial meaning (Sasiekharan, Ph.D.Thesis R.1991, Havard University; Sasiekharan; R.et al.; A comparative analysis of the primary sequences and characteristics of heparinase I; II and III from Flavobacterium heparinum Biochemical and Biophysical Research Communication 1996 Vol.229:770-777): heparinase is a kind of of polysaceharide lyase; Be used to study the interaction between heparinase and the substrate polysaccharide heparin thereof, help to illustrate the mechanism of action of polysaceharide lyase; Heparinase can be used to resolve the structure and the biological function thereof of complicated mucopolysaccharides such as heparin; Heparinase can be used to resolve intravital blood coagulation of people and anticlotting mechanism; Heparinase can prevent postoperative hemorrhage as the removal of clinical blood heparinization; Heparinase is used for the processing of the preceding blood products of PCR reaction; Heparinase can be used to prepare low molecular anticoagulation medicine low molecular weight heparin.
Heparinase from the heparin Flavobacterium mainly contains three kinds; Difference called after Heparinase I (EC 4.2.2.7), Heparinase I I (No EC code) and Heparinase I II (EC 4.2.2.8) (Robert J.Linhardt et al., Purification and characterization of heparin lyases from Flavobacterium heparinum JBC 1992Vol.267:24347-24355).Heparinase I mainly acts on heparin, molecular weight 43kDa, and Heparinase I I acts on heparin and Suleparoid, molecular weight 85kDa, Heparinase I II mainly acts on Suleparoid, molecular weight 73kDa.Wherein, more to the research of Heparinase I, and less relatively to the research of Heparinase I I and III.
HSPG is a kind of baroque acidic protein glycan; Participate in a series of physiology and pathologic process in the body; Like (Kjellen L such as fetal development, neurite outgrowth, vascularization, tissue repair, inflammation, autoimmunization, tumor growth and transfers; Lindahl U.Proteoglycans:Structures and interactions Annu Rev Biochem, 1991 Vol.60:440-443; Bernfield M, Gotte M, Park P W, et al.Functions of cell surface heparan sulkfate proteoglycans Annu Rev Biochem, 1999,68:726-729).The enzymolysis of Suleparoid is its principal element of bringing into play above-mentioned effect.Can discharge and activate various behind the enzymolysis and its bonded bioactive molecule, thereby influence various physiology and pathologic process.Therefore, the enzyme of degraded Suleparoid is vital regulatory factor, studies this enzyme (Heparinase I I) and has important significance for theories and using value.The acquisition yet Heparinase I I normally purifies from heparin Flavobacterium fermented liquid; When producing Heparinase I I, the heparin Flavobacterium produces Heparinase I, III and four kinds of chondroitinases (chondroitinase B, C, ABC; AC); Make the separation and purification of Heparinase I I become complicated, need the chromatogram purification through multistep usually, yield is very low.Heparin Flavobacterium rate of growth is slow; The production stability of heparinase is poor, and producing heparinase needs expensive heparin-induced; Increased the cost of enzyme, thus the allos of Heparinase I I recombinant expressed be to substitute the effective and feasible scheme that the heparin Flavobacterium is produced Heparinase I I.
Yet very limited to the recombinant expressed research of the allos of Heparinase I I, up to the present, bibliographical information has only that Fu Wen is refined to be studied it with people such as Su.People such as Fu Wenbin utilize the pET expression system to realize Heparinase I I recombinant expressed in intestinal bacteria, and the SDS-PAGE electrophoresis result shows the target protein band, but expression amount is very low, and form inclusion body easily, and solubility heparin enzymic activity is merely 50U/L A
600(Fu Wenbin, Yu Xiao etc., the cloning and expression of flavobacterium heparinum enzyme II, food and medicine, 2007, Vol.9:1-4); Expressed Heparinase I I activity increases in the Su research, but needs the multistep purifying, causes the purifying expense than higher; Be unfavorable for industrial applications (Su H, blain F, Musil RA; Zimmermann JJF, Gu K, Bennett DC.Isolation and Expression in Escherichia coli of hepB and hepC; Genes Coding for the Glycosaminoglycan-Degrading Enzymes Heparinase II and Heparinase III; Respectively, from Flavobacterium heparinum.Appl.Environ.Microbiol.1996,62:2723-2734).Discover, can significantly improve the recombinant expressed solubility of enzyme through fusion with maltose binding protein (MBP) and enzyme.This research group utilizes fusion protein technology that nitrogen end and the low temperature induction that MBP is fused to Heparinase I improved the recombinant expressed solubility of Heparinase I significantly.This work is applied for a patent, and the patent No. is 200410038098.6.And from colibacillary natural MBP can with the special absorption of SANMALT-S, participate in transhipment and the utilization of intestinal bacteria to SANMALT-S.MBP not only can combine with amylose starch; Realize affine separation; Also can realize affine the separation (Usha Srinivasan et al., A convenient method for affinity purification of maltose binding protein fusions.Journal of Biotechnology 1998 Vol with yam starch.62:163-167; Lian Dejun etc., a kind of improved fusion rotein affinity chromatographic purification process, biological chemistry and biological progress 1998 Vol.25:283-284), thus the separation and purification cost of enzyme can be reduced greatly, help realizing the separation and purification of industrially scalable.
Summary of the invention
The object of the present invention is to provide a kind of Heparinase I I fusion rotein and encoding sox thereof.
Fusion rotein provided by the invention (called after MBP-HepB), be following a) or b) albumen:
A) protein that the aminoacid sequence shown in the 1-1118 of sequence 2 is formed in the sequence table;
B) in sequence table the aminoacid sequence of sequence 2 through replacing and/or disappearance and/or add one or several amino acid and have a Heparinase I I active by a) deutero-protein.
The aminoacid sequence of MBP among this fusion rotein MBP-HepB as SEQ ID No:2 from shown in the amino acid 1-367; The aminoacid sequence of HepB in this fusion rotein as SEQ ID No:2 from shown in the amino acid 372-1118.
The encoding sox of above-mentioned fusion rotein also belongs within protection scope of the present invention.
Further, said gene is following 1) or 2) or 3):
1) encoding sequence is a sequence 1 in the sequence table;
2) under the rigorous condition of height with sequence table in the nucleotide sequence hybridization and the coding claim 1 said proteic nucleotide sequence of sequence 1;
3) with sequence table in the nucleotide sequence shown in the sequence 1 have the homology 95% or more and the said proteic nucleotide sequence of claim 1 of encoding.
In the sequence 1, the coding region of MBP is the base from 5 ' end 1-1101 position of sequence 1; The coding region of HepB is the base from 5 ' end 1114-3360 position of sequence 1.
The rigorous condition of said height is meant, with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.2,7%SDS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 30min for 65 ℃ to add film washing liquid II.
The recombinant vectors or the transgenic cell line that contain said gene also belong within protection scope of the present invention.
Further, above-mentioned recombinant vectors makes up through following steps:
MCS with the insertion of the dna fragmentation shown in the sequence in the sequence table 1 plasmid pMAL-c2x obtains recombinant expression vector.
The reorganization bacterium that contains said gene also belongs within protection scope of the present invention.
Further, above-mentioned reorganization bacterium is the recombination bacillus coli that contains above-mentioned recombinant vectors.
The host of above-mentioned recombination bacillus coli is TB1 preferably.
Another object of the present invention is to provide a kind of method of producing Heparinase I I.
Method provided by the invention is with above-mentioned reorganization bacterium inducing culture, expresses to obtain Heparinase I I.
Above-mentioned inducing culture condition is: the IPTG of 0.01-1mM, 10-42 ℃ inducing culture 15-28 hour; The IPTG of 0.24mM preferably, 15 ℃ of inducing culture 24 hours.
The solvent of the substratum of above-mentioned inducing culture is a water, and solute is 10g/L NaCl, 5g/L yeast extract, 10g/L peptone, 1% (mass percent) ethanol, 0.6mg/L paraxin and 100 μ g/L penbritins.
The present invention has made up the fusion expression vector that prevents that inclusion body from forming, and realizes an affine separation of step through the amylose resin.Realize first that through fusion rotein Heparinase I I has activity, correct folding soluble protein form to exist more than 90% in intestinal bacteria; Intestinal bacteria TB1 (pMAL-hepB) 37 degrees centigrade cultivate 2.5 hours after; Add 0.24mM IPTG, 15 degrees centigrade of inducing temperatures, yeast powder concentration 0.5% in the substratum; Ethanol 1%; The Heparinase I I enzyme work of producing can reach the 118.4IU/L fermented liquid, and expression amount can reach the 179mg/L fermented liquid, can reach 0.66IU/mg than enzyme work.The present invention also can realize the single step purification of this fusion rotein through affine separation.The present invention adds the IPTG time through changing IPTG concentration, inducing temperature, optimization and improvements such as medium component the output and heparinase work of fusion rotein.The present invention will play a significant role in the production of Heparinase I I.
Description of drawings
Fig. 1 is the building process synoptic diagram of expression vector pMAL-hepB.
Fig. 2 is the Heparinase I I gene electrophoretogram that pcr amplification obtains from the heparin Flavobacterium.
Fig. 3 is transformant PCR checking electrophoretogram.
Fig. 4 cuts the checking electrophoretogram for the transformant enzyme.
Fig. 5 is a fusion plasmid pMAL-hepB host optimum result: (a) be that OD600, crude enzyme liquid enzyme are lived and protein concentration compares; (b) be that crude enzyme liquid is more alive relatively than enzyme.
Fig. 6 is the SDS-PAGE figure that intestinal bacteria TB1/pMAL-hepB engineering strain is expressed MBP-HepB.
Fig. 7 passes through the affine isolating SDS-PAGE electrophoretogram of amylose resin for fusion rotein MBP-HEPB.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.
Among the following embodiment,, be ordinary method like no specified otherwise.Said primer synthesizes and examining order is accomplished by Invitrogen Bioisystech Co., Ltd; All restriction enzymes are all available from New England Biolabs company; Pfu enzyme and dNTP are available from TaKaRa company; All competent cells (as: DH5 α, TB1 and Top10) are available from the Beijing Quanshijin Biotechnology Co., Ltd; Heparinase I I enzyme activity determination substrate heparin is available from the biochemical medicine company of Hebei Changshan limited-liability company, and other pharmaceutical chemicals is the general analysis pure reagent, available from Chemical Reagent Co., Ltd., Sinopharm Group.
The expression of embodiment 1, Heparinase I I fusion rotein MBP-HepB
One, removes the clone of the heparin flavobacterium heparinum enzyme II encoding sequence of signal peptide
The building process of expression vector pMAL-hepB is as shown in Figure 1, and detailed process is following:
1, primer design and synthetic
Obtain dna sequence dna (Su, H., the Blain of heparin Flavobacterium (Flavobacterium heparinum) Heparinase I I through the Genbank inquiry; F., Musil, R.A.; Zimmermann, J.J., Gu; K.and Bennett, D.C.Isolation and expression in Escherichia coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III; Respectively, from Flavobacterium heparinum.Appl.Environ.Microbiol.1996,62; 2723-2734); Dna sequence dna according to the heparin flavobacterium heparinum enzyme II that removes coded signal peptide base designs primer again, and in primer sequence, introduces the recognition site of restriction enzyme Sac I and Pst I, and used upstream and downstream primer is respectively:
Upstream primer P1:5 '-ATAT
GAGCTCGCAAACCAAGGCCGATGTG-3 ' (base of band underscore is the restriction enzyme site of SacI),
Downstream primer P2:5 '-
CATACTGCAGTCATTATCTCAAAAAACGGTAGGTTCCTTC-3 ' (base of band underscore is the restriction enzyme site of PstI) after the amplification, promptly introduces Sac I and Pst I restriction enzyme site respectively.
2, pcr amplification is removed the encoding sequence of the heparin flavobacterium heparinum enzyme II of signal peptide
The reaction system of pcr amplification is: 50ng heparin Flavobacterium genomic dna masterplate, every kind of primer of 100pmol, 1 * amplification buffer (sky, Beijing is a Bioisystech Co., Ltd), every kind of dNTP of 200 μ mol/L, the 1 high-fidelity Pfu of unit enzyme; Amplification program is: 95 degrees centigrade of sex change 5 minutes, and 50,52,54,56,58 or 60 degrees centigrade of primer annealings 1 minute, 72 degrees centigrade were extended 3 minutes, and after 30 circulations, 72 degrees centigrade are extended and finished reaction in 10 minutes.This PCR result is as shown in Figure 2, showing that amplification has obtained the Heparinase I I gene fragment of 2.2kb, and the nucleotide sequence that order-checking shows product is shown in the 1114th-3360 of sequence in the sequence table 1 (called after HepB).Among Fig. 2; It is 60,58,56,54,52 or 50 degrees centigrade of amplifications that swimming lane 1-6 is respectively annealing temperature; Swimming lane M is molecular weight marker (stripe size is followed successively by 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.5kb, 1kb, 800bp, 500bp, 300bp), and arrow indication place is the 2.2kb target fragment.
3, structure contains the segmental cloning vector of purpose
Reference reagent box specification sheets is operated, and the direct subclone of purpose fragment of the pcr amplification of step 2 in the step 1 is gone into carrier pMD
TMAmong-19T the Simple (TaKaRa company), obtain connecting product.
4, the screening and the order-checking of transformed into escherichia coli and positive colony transformant
With the connection product transformed into escherichia coli DH5 α competent cell that step 3 obtains, concrete grammar is: with the connection product of 10 μ l and the bacillus coli DH 5 alpha competent cell mixing of 100 μ l, ice bath 30 minutes; 42 degrees centigrade of heat shocks 90 seconds, ice bath 10 minutes adds LB liquid nutrient medium (the peptone 3g that 800 μ l contain 100 μ g/L penbritins then; Yeast extract 1.5g, NaCl 3g, water 285mL) in; 180rpm, 37 degrees centigrade of joltings 60 minutes are applied to the LB resistance culture plate (peptone 3g, the yeast extract 1.5g that contain 100 μ g/L penbritins; NaCl 3g; Agar powder 4.5g, water 285mL, 16 μ l X-gal and 4 μ l IPTG/ are dull and stereotyped) carry out blue hickie screening.Cultivated 12-20 hour for 37 degrees centigrade.Select hickie and as masterplate, carry out bacterium colony PCR evaluation with primer P1 and P2, PCR reaction system and reaction conditions are identical with step 2.After reaction finishes, amplified production is carried out 0.8% agarose gel electrophoresis detect, can contain the positive colony of transformant.The positive colony that screening is obtained goes to 5mL and contains in the LB liquid nutrient medium of 0.05mg/mL penbritin; 37 degrees centigrade, 220rpm jolting 12 hours; Bacterium liquid is transferred to invitrogen Bioisystech Co., Ltd check order, will contain the pMD of the Heparinase I I protein coding gene of 1114-3360 position nucleotide sequence with sequence 1 in the sequence table
TM-19T Simple recombinant vectors called after pMD-19-hepB.
Two, the structure of expression of recombinant e. coli carrier
With the pMD-19-hepB plasmid is masterplate, carries out the gene of pcr amplification heparin flavobacterium heparinum enzyme II with primer P1 and P2, and 2 is identical in PCR reaction system and reaction conditions and the step 1.PMAL-c2x carrier (available from U.S. New England Biolabs company) and PCR product (is the amplified production of masterplate with the pMD-19-hepB plasmid) are used Sac I and Pst I double digestion respectively, use T
4Dna ligase (TaKaRa company) connects; Transforming DH5 α, is primer with P1 and P2, through bacterium colony PCR screening transformant (as shown in Figure 3); Extraction can obtain the pMAL-c2x recombinant vectors in the transformant of 2.2kb PCR product, verifies (as shown in Figure 4) through Sac I with Pst I double digestion respectively.M is molecular weight marker (stripe size is followed successively by 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.5kb, 1kb, 800bp, 500bp, 300bp) among Fig. 3; Swimming lane 1,2 is the transformant of PCR checking, and arrow indication place is a Heparinase I I gene band.M is molecular weight marker (stripe size is followed successively by 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1.5kb, 1kb, 800bp, 500bp, 300bp) among Fig. 4; Swimming lane 1,2 is recombinant plasmid pMAL-hepB double digestion rear electrophoresis figure; Arrow indication place is a Heparinase I I gene band; Swimming lane 3,4 is the correct recombinant plasmid pMAL-hepB that connects, and swimming lane 5,6 is original plasmid pMAL-c2x.To check order through the segmental plasmid of 2.2kb that Sac I and Pst I double digestion obtain, will contain the pMAL-c2x recombinant vectors called after pMAL-hepB of the Heparinase I I fusion rotein encoding sox of 1114-3360 position nucleotide sequence with sequence 1 in the sequence table.In pMAL-hepB, two successive termination codon TAATGA are arranged between hepB gene and the lacZ α gene, can not express lacZ α albumen thereby can stop protein translation effectively.
Three, the expression of Heparinase I I fusion rotein MBP-HepB
Contain the plasmid in the bacillus coli DH 5 alpha of pMAL-hepB in the extraction step two, (plysS) according to ordinary method transformed into escherichia coli TB1, Top10, JM109, BL21 (DE3), BL21 (DE3).Except that E.coli TB1 competent cell is made the explanation for preparing competent escherichia coli cell according to Calcium Chloride Method in " molecular cloning experiment guide " accomplishes, E.coliTOP10, E.coli DH5 α, E.coli JM109, E.coli BL21 (DE3), E.coli BL21 (DE3) (plysS) competent cell all available from the Beijing Quanshijin Biotechnology Co., Ltd.The process penbritin screens and utilizes the primer that step 1 provides in the step 1 to carry out bacterium colony PCR evaluation; The intestinal bacteria TB1, Top10, JM109, BL21 (DE3) and the BL21 (DE3) that obtain containing pMAL-hepB (plysS), promptly TB1/pMAL-hepB, Top10/pMAL-hepB, JM109/pMAL-hepB, BL21 (DE3)/pMAL-hepB, BL21 (DE3) (plysS)/pMAL-hepB and DH5 α/pMAL-hepB be as the engineering bacteria of expressing MBP-HepB.
With plasmid pMAL-c2x transformed into escherichia coli TB1, Top10, JM109, BL21 (DE3), BL21 (DE3) (plysS) with DH5 α, obtain empty carrier contrast TB1/pMAL-c2x, Top10/pMAL-c2x,, JM109/pMAL-c2x, BL21 (DE3)/pMAL-c2x, BL21 (DE3) (plysS)/pMAL-c2x and DH5 α/pMAL-c2x.
Below operation is carried out top engineering bacteria is parallel.
Empty carrier contrast and engineering bacteria are being contained LB substratum (the NaCl 10g/L of amicillin resistance respectively; Yeast extract is 5g/L; Peptone 10g/L, 1% (mass percent) ethanol, 0.6mg/L paraxin; Contain 100 μ g/L penbritins) 37 degrees centigrade cultivate after 2.5 hours, adding final concentration is that 15 degrees centigrade of 0.24mM IPTG induced 24 hours.10000rpm, 8 minutes centrifugal collection thalline are also with 20mmol/L Tris-HCl (pH 7.5) washed twice, and are resuspended to OD
600Be about near 8.000.With top OD
600Be about 8.000 resuspended liquid and carry out ultrasonication (output rating is 300W, each ultrasonic 3 seconds with intermittently 3 seconds processing 198 times), 12000rpm, 30 minutes are centrifugal, and the supernatant of centrifugal gained is crude enzyme liquid after the ultrasonication.The optical absorption method of 232nm is adopted in the detection of enzyme activity (unit is IU/L), and the enzyme work of 1IU is defined as the reaction effectiveness that 30 centigrade per minutes produce 1 μ mol unsaturated link(age).Taking heparin substrate solution 0.5ml (25g/L heparin, 40mM NaCl, 3.5mM CaCl
2, 17mM Tris-HCl, pH 7.5), the crude enzyme liquid of gained in the step in the adding, other volumes replenish with the Tris damping fluid, and final reaction volume is 1.5ml, surveys the absorbancy changes delta A of inherent 232nm of unit time
232Extinction coefficient epsilon=3800M
-1The ratio that is defined as enzyme activity and crude enzyme liquid protein concentration (unit is mg/L) than enzyme (unit is an IU/mg albumen) alive.Conventional Bradford method is adopted in the protein concentration monitoring.
The result shows: empty carrier control strain TB1/pMAL-c2x, Top10/pMAL-c2x, JM109/pMAL-c2x, BL21 (DE3)/pMAL-c2x, BL21 (DE3) (plysS)/pMAL-c2x and DH5 α/pMAL-c2x inducing culture after no enzyme live; The Other Engineering bacterium has all given expression to the MBP-HepB fusion rotein of solubility; Wherein the expression effect in intestinal bacteria TB1 is best; The result is as shown in Figure 5: no matter live or than enzyme work from OD value, the enzyme of bacterium, TB1 all be better than other host (TB1 OD value during, enzyme work for the host and than enzyme work be respectively 3.87,118.24IU/L and 0.66IU/mg).And through order-checking, TB1/pMAL-hepB, Top10/pMAL-hepB, JM109/pMAL-hepB, BL21 (DE3)/pMAL-hepB, BL21 (DE3) (plysS)/ proteic aminoacid sequence that pMAL-hepB and DH5 α/pMAL-hepB give expression to is all shown in sequence table SEQ ID No:2; And the aminoacid sequence of the MBP of this fusion rotein MBP-HepB as SEQ ID No:2 from shown in the amino acid 1-367; The aminoacid sequence of the HepB of this fusion rotein MBP-HepB as SEQ ID No:2 from shown in the amino acid 372-1118.
Best host TB1 expressed proteins is carried out the SDS-PAGE electrophoresis: supernatant (crude enzyme liquid) the 40 μ l of centrifugal gained do soluble protein component S DS-PAGE electrophoresis after getting the step ultrasonication, get after the above-mentioned ultrasonication deposition of centrifugal gained and do soluble protein ingredient SDS-PAGE electrophoresis.The result is as shown in Figure 6; M is marker (from top to bottom molecular weight is 230kDa, 150kDa, 100kDa, 80kDa (redness), 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, 15kDa, 10kDa successively) among the figure, and 1,2 are respectively soluble protein ingredient and the soluble protein component of empty carrier contrast TB1/pMAL-c2x; 3,4 be respectively intestinal bacteria TB1/pMAL-hepB soluble protein ingredient and soluble protein component, arrow indication place is fusion rotein MBP-HepB (123KDa).
Fusion partners (fusion partner) the maltose binding protein MBP that the present invention utilizes can separate with affine one step of absorption realization of amylose starch.Concrete affine separating step is following: with final concentration is 24 hours thalline 100mL of 0.24mM IPTG abduction delivering, centrifugal 5 minutes of 10000rpm; Establish the not thalline contrast of abduction delivering simultaneously.Then operate respectively by following two schemes:
Scheme one: with column equilibration liquid Column buffer (20mM Tris-HCl; 200mM NaCl, pH7.5) washed twice is resuspended among the 5mL Column buffer; Carry out ultrasonication (output rating is 300W, each ultrasonic 3 seconds with intermittently 3 seconds processing 198 times).
Scheme two: osmotic pressure impacts.Thalline was resuspended among the 100mL osmotic shock buffer I (20-40% sucrose, 30mM Tris-HCl, 1mM EDTA) 15 minutes, stirs.Centrifugal 10 minutes of 10000rpm is resuspended in the equal-volume 0.5mM sal epsom then, and ice bath 10-15 minute, 10000rpm, centrifugal 10 minutes.
Centrifugal back supernatant is with the amylose starch affine separator column of 0.5ml/min through the 2ml pre-equilibration, through 10mM0.5ml/min SANMALT-S wash-out and collection.
Each step gets 40 μ l and is SDS-PAGE, after target protein process amylose starch (amylose) resin absorption, and can be under 1 column volume with 10mM SANMALT-S with the target protein wash-out.The result is as shown in Figure 7, shows through target protein behind the amylose resin single step purification to account for more than 95%.Among Fig. 7; M is marker (from top to bottom molecular weight is 230kDa, 150kDa, 100kDa, 80kDa (redness), 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, 15kDa, 10kDa successively); 1 is the soluble protein component of 0.24mM IPTG inductive intestinal bacteria TB1 (pMAL-hepB), 2,3; 4; 5,6 are respectively 0.24mM IPTG inductive intestinal bacteria TB1 (pMAL-hepB) through the protein ingredient of amylose resin absorption back with the different concns of SANMALT-S wash-out collection, and the arrow indication is a target protein.
Claims (11)
1. albumen is following albumen a):
A) protein that the aminoacid sequence shown in the 1-1118 of sequence 2 is formed in the sequence table.
2. the said proteic encoding sox of claim 1.
3. gene according to claim 2 is characterized in that: said gene is following 1):
1) encoding sequence is a sequence 1 in the sequence table.
4. the recombinant vectors or the transgenic cell line that contain claim 2 or 3 said genes.
5. recombinant vectors according to claim 4 is characterized in that: said recombinant vectors makes up through following steps:
MCS with the insertion of the dna fragmentation shown in the sequence in the sequence table 1 plasmid pMAL-c2x obtains recombinant expression vector.
6. the reorganization bacterium that contains claim 2 or 3 said genes.
7. reorganization bacterium according to claim 6 is characterized in that: said reorganization bacterium is the recombination bacillus coli that contains claim 4 or 5 described recombinant vectorss.
8. a method of producing heparinase is with claim 6 or 7 described reorganization bacterium inducing culture, expresses to obtain heparinase.
9. method according to claim 8 is characterized in that: said inducing culture condition is: the IPTG of 0.01-1mM, 10-42 ℃ inducing culture 15-28 hour.
10. method according to claim 9 is characterized in that: said inducing culture condition is: the IPTG of 0.24mM, 15 ℃ of inducing culture 24 hours.
11. according to claim 9 or 10 described methods, it is characterized in that: the solvent of the substratum of said inducing culture is a water, solute is 10g/L NaCl; The 5g/L yeast extract; 10g/L peptone, 1% (mass percent) ethanol, 0.6mg/L paraxin and 100 μ g/L penbritins.
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| CN103103174B (en) * | 2011-11-11 | 2014-07-23 | 清华大学 | Chondrosulphatase AC fusion protein, and coding gene and construction method thereof |
| CN102888391B (en) * | 2012-10-30 | 2015-01-21 | 深圳市海普瑞药业股份有限公司 | Immobilization method of heparinase II |
| CN105753945B (en) * | 2014-12-15 | 2019-04-09 | 清华大学无锡应用技术研究院 | A kind of link peptide and its application |
| CN105754979B (en) * | 2014-12-15 | 2020-03-17 | 清华大学无锡应用技术研究院 | MBP fusion heparinase II and coding gene and preparation method thereof |
| CN111218466A (en) * | 2019-11-25 | 2020-06-02 | 深圳大学 | Fusion gene MBP-H1 for expressing heparinase and recombinant plasmid and application thereof |
| CN113493780A (en) * | 2021-01-25 | 2021-10-12 | 上海宝维医药技术有限公司 | Method for preparing recombinant heparinase II by utilizing SUMO fusion expression system and SUMO _ heparinase II fusion protein prepared by same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699424A (en) * | 2004-05-19 | 2005-11-23 | 清华大学 | Heparinase I fusion protein and genes encoding same and expression method thereof |
| CN101608178A (en) * | 2009-07-29 | 2009-12-23 | 清华大学 | Fusion heparinase and encoding gene thereof and preparation method |
| CN101608179A (en) * | 2009-07-29 | 2009-12-23 | 清华大学 | Fusion heparinase and encoding gene thereof |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699424A (en) * | 2004-05-19 | 2005-11-23 | 清华大学 | Heparinase I fusion protein and genes encoding same and expression method thereof |
| CN101608178A (en) * | 2009-07-29 | 2009-12-23 | 清华大学 | Fusion heparinase and encoding gene thereof and preparation method |
| CN101608179A (en) * | 2009-07-29 | 2009-12-23 | 清华大学 | Fusion heparinase and encoding gene thereof |
Non-Patent Citations (1)
| Title |
|---|
| 叶逢春等.重组大肠杆菌生产可溶性MBP融合肝素酶的培养条件优化.《生物加工过程》.2006,第4卷(第3期),28-32. * |
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