CN1312183C - Heparinase I fusion protein and genes encoding same and expression method thereof - Google Patents
Heparinase I fusion protein and genes encoding same and expression method thereof Download PDFInfo
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- CN1312183C CN1312183C CNB2004100380986A CN200410038098A CN1312183C CN 1312183 C CN1312183 C CN 1312183C CN B2004100380986 A CNB2004100380986 A CN B2004100380986A CN 200410038098 A CN200410038098 A CN 200410038098A CN 1312183 C CN1312183 C CN 1312183C
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- Prior art keywords
- heparinase
- fusion rotein
- seq
- lys
- ala
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Abstract
The present invention discloses heparinase I fusion protein and encoding genes and an expression method thereof. The heparinase I fusion protein provided by the present invention is protein having an amino acid residue sequence of SEQ ID No. 2 in a sequence table or protein which has the same activity as that of the amino acid residue sequence of the SEQ ID No. 2, is derived from the SEQ ID No. 2 and is formed by means that the amino acid residue sequence of the SEQ ID No. 2 is replaced, deleted or added by one or a plurality of amino acid residues. The present invention is characterized in that more than 90% of the heparinase exists in escherichia coli in the modes of activity and correct folded soluble protein. The heparinase activity can achieve 270 U/L in fermentation liquid (108UL <-1> OD<600><-1>), and the content of the protein can achieve 150 mg/L. In addition, the single step purification of the fusion protein is realized by compatible separation. The present invention can be widely used for producing the heparinase I.
Description
Technical field
The present invention relates to a kind of Heparinase I fusion rotein and encoding gene and expression method in genetically engineered and the field of fermentation engineering.
Background technology
Heparinase (heparinase) is the polysaceharide lyase that a class acts on heparin, in many kinds of microorganisms, find, comprise excellent bacillus Corynebacterium sp. (high Ningguo etc., heparinase produces screening and the fermentation condition of bacterium, microorganism journal 1999 Vol.39:64-67), Sphingobacterium sp. Sphingobacterium sp. (high Ningguo etc., the generation of Sphingobacterium sp. heparinase, microorganism journal 2003 Vol.43:813-816), Bacillus subtillis Bacillus subtilis (Wang Zhongyan etc., heparinase produces the screening of bacterium and the research of thick enzymatic property thereof, Sichuan University's journal (natural science edition) 2002 Vol.39:777-779), Bacillus circulans Bacillus circulans (Yasutaka Tahara et al., Purification and characterization of heparinasethat degrades both heparin and heparin sulfate from Bacillus circulansBioSci.Biotechnol.Biochem.2002 Vol.66:1181-1184), Bacteroides heparinolyticus Prevotellaheparinolytica (Kazuyuki Sugahara et al., Characterization of heparinasefrom an oral bacterium Prevotella heparinolytica J.Biochem.1998Vol.123:283-288), Bacteroides stercoris Bacteroides stercoris HJ-15 (Dong Hyuu Kim etal., Purification and characterization of a novel heparinase from Bacteroidesstercoris HJ-15 J.Biochem.2000 Vol.128:323-328) and heparin Flavobacterium Flavabacteriumheparinum (Sasiekharan, R.1991 Ph.D.Thesis, Havard University).But the heparinase from the heparin Flavobacterium is business-like unique source.Heparinase from the heparin Flavobacterium mainly contains three kinds, difference called after Heparinase I (EC 4.2.2.7), II (NO EC code) and III (EC 4.2.2.8) (RobertJ.Linhardt et al., Purification and characterization of heparin lyases fromFlavobacterium heparinum JBC 1992 Vol.267:24347-24355).
The research of heparinase has crucial meaning (Sasiekharan, Ph.D.Thesis R.1991, HavardUniversity; Sasiekharan, R.et al., A comparative analysis of the primarysequences and characteristics of heparinase I, II, and III from Flavobacteriumheparinum Biochemical and Biophysical Research Communication 1996Vol.229:770-777): heparinase is a kind of of polysaceharide lyase, is used to study the mechanism of action that interaction between heparinase and the substrate polysaccharide heparin thereof helps to illustrate 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 be used to prepare low molecular anticoagulation medicine Low molecular heparin; 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.
Normally purification obtains Heparinase I from heparin Flavobacterium fermented liquid, needs the chromatogram purification through multistep usually, and yield is lower.Heparin Flavobacterium rate of growth is slow, and the production stability of heparinase is poor, and producing heparinase need be expensive heparin-induced, has increased the cost of enzyme.Therefore, utilize the cost of Flavobacterium production heparinase high at present, limited the application development of heparinase.Ram Sasiekharan (1993) (Sasiekharan, R.et al., Cloning and expression of heparinase I gene fromFlavobacterium heparinum Proc.Natl.Acad.Sci.USA 1993 Vol.90:3660-3664) at first by amino acid sequencing, the design primer amplification has obtained the gene order of Heparinase I and has been binned in the expression (table 1) that has realized heparinase in the intestinal bacteria; But expression product can not correctly be folded to form activated albumen.RamSasiekharan (1996) (Sasiekharan, R.et al., Expression in Escherichia coli, Purification and characterization of heparinase I from Flavobacteriumheparinum Biochem.J.1996 Vol.315:589-597) utilize the pET expression system to improve expressing quantity (14.4mg/L fermented liquid) (table 1), but the reorganization Heparinase I still forms inclusion body, needs renaturation could form activated protein.Oded Shoseyov (1999) (Etai Shpigel, et al., Immobilizationof recombinant heparinase I fused to cellulose-binding domain Biotechnologyand Bioengineering 1999 Vol.65:17-23) utilize Mierocrystalline cellulose conjugated protein (CBD) and heparinase to merge, utilize fusion rotein to realize the amalgamation and expression of CBD-hep, hep-CBD, by the cellulose column affinity separations, and improved the expression amount (150mg/L fermented liquid) (table 1) of Heparinase I; Although but under low IPTG (0.5mM), to induce, this fusion rotein still forms inclusion body, needs renaturation just can be formed with active reorganization Heparinase I.
The recombinant expressed present Research of table 1. Heparinase I
| Recombinant expressed research | Expression amount (mg/L fermented liquid) | Host, carrier | Whether solvable | Whether secrete | Whether be fusion rotein |
| Ram Sasiekharan (1993) Ram Sasiekharan (1996) | 0.1 14.4 | E.coli BL21(DE3) (pET3a-hepA) E.coli BL21(DE3) (pET3a、pET12a、pET15b、 pET28a-hepA) | Small part is solvable not | Not | Not |
| Oded Shoseyov (1999) | 150 | E.coli BL21(DE3) (pET3d CBD-hepA， pET3d hepA-CBD) | Not | Not | CBD fusion protein |
From colibacillary natural maltose binding protein MBP can with the special absorption of maltose, participate in transhipment and the utilization of intestinal bacteria to maltose.MBP not only can combine with amylose, realizes affine separation, also can with yam starch in conjunction with realizing affine the separation that (biological chemistry and biophysics are made progress 1998 Vol.25:283-284 for Lian Dejun etc., a kind of improved fusion rotein affinity chromatographic purification process; Usha Srinivasan et al., Aconvenient method for affinity purification of maltose binding proteinfusions Journal of Biotechnology 1998 Vol.62:163-167), thus can reduce the cost of the separation and purification of enzyme greatly.
The innovation and creation content
The purpose of this invention is to provide a kind of Heparinase I fusion rotein and encoding gene thereof.
Heparinase I fusion rotein provided by the present invention, name is called MBP-HepA, be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.
SEQ ID № in the sequence table: 2 are made up of 756 nitrogen bases acid residues.
Heparinase I fusion rotein encoding gene, name is called MBP-HepA, has one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 95% above homology, and the identical function protein DNA sequence of encoding.
Dna sequence dna in the sequence 1 is by 2271 based compositions, and the open reading frame of this gene is from 5 ' end the 1st to the 2271st bit base.
Contain expression carrier of the present invention, clone and engineering bacteria, all belong to protection scope of the present invention as pMal-hepA and the intestinal bacteria E.coli TB1 (pMAL-hepA) that contains pMal-hepA.
Increase arbitrary segmental primer in this fusion rotein encoding gene to also within protection scope of the present invention.
Second purpose of the present invention provides a kind of method of expressing the Heparinase I fusion rotein.
The method of expression Heparinase I fusion rotein provided by the present invention is that the recombinant expression vector that will contain above-mentioned Heparinase I fusion rotein encoding gene imports the expressive host bacterium, expresses the Heparinase I fusion rotein.
Wherein, the recombinant expression vector of described Heparinase I fusion rotein encoding gene is pMal-hepA.
PMal-hepA inserts pMal p2x by BamHI and PstI with Heparinase I encoding gene complete sequence, the recombinant expression vector of the Heparinase I fusion rotein encoding gene that obtains in pMalc2x (physical map is bought as shown in Figure 7 from the NEB company) expression vector.Wherein, described Heparinase I encoding gene complete sequence is that the genome DNA with the heparin Flavobacterium is a template, with 5 ' GCCTGGATCCCAGCAAAAAAAATCCGGTAAC3 ' and 5 ' GCTTCTGCAGTCTGGCAGTTTCGCTGTAC3 ' is primer, obtains according to conventional PCR method.
Described host bacterium can be intestinal bacteria, and described intestinal bacteria can be following bacterial strain: B121, JM109, DH5 α, TB1 etc.
The abduction delivering condition of Heparinase I fusion rotein can be 10-42 degree centigrade of 0.01-1mM IPTG and induced 16 hours; Yeast extract concentration in the substratum that is adopted can be 1g/L-20g/L, also can add 1%-5% ethanol and/or heat-shocked materials such as 0.05-2mg/L kantlex and/or 0.01-2.0mg/L paraxin in substratum.Wherein, this expression condition be preferably engineering bacteria 37 degrees centigrade cultivate 3 hours after, add 0.3mM IPTG and induced 16 hours for 15 degrees centigrade: the substratum that is adopted is preferably NaCl 10g/L, yeast extract paste is 7.5g/L, peptone 10g/L, 1% (mass percent) ethanol, 0.6mg/L paraxin.
The present invention has made up the fusion expression vector that prevents that inclusion body from forming, and realizes an affine separation of step by the amylose resin.Realize first that by fusion rotein Heparinase I exists with the soluble protein form that activity is arranged, correctly fold more than 90% in intestinal bacteria; Intestinal bacteria E.coli TB1 (pMAL-hepA) adds 0.3mM IPTG 37 degrees centigrade of cultivations after 3 hours, 15 degrees centigrade of inducing temperatures, and yeast powder concentration 0.75% in the substratum, ethanol 1%, the heparinase enzyme work of production can reach 270U/L (108 UL
-1OD600
-1) fermented liquid, expression amount can reach the 150mg/L fermented liquid, and has realized the single step purification of this fusion rotein by affine separation.The present invention adds the IPTG time by changing IPTG concentration, inducing temperature, optimization and improvements such as substratum composition warm proteic output and heparinase live.The present invention will play a significant role in the production of Heparinase I.
Description of drawings
Fig. 1 is the building process synoptic diagram of expression vector pMal-hepA
Fig. 2 is the Heparinase I gene electrophoretogram that pcr amplification obtains from the heparin Flavobacterium
Fig. 3 is transformant bacterium colony PCR and double digestion checking electrophoretogram
Fig. 4 a is the protein electrophorese collection of illustrative plates of E.coli TB1 (pMAL-hepA) at 37 ℃ of different induction times
Fig. 4 b is the protein electrophorese collection of illustrative plates of E.coli TB1 (pMAL-hepA) at 21 ℃ of different induction times
Fig. 5 passes through the affine isolating SDS-PAGE electrophoretogram of amylose resin for fusion rotein MBP-HEPA
Fig. 6 is the physical map of pMal-hepA
Fig. 7 is pMal p2x, the physical map of pMal c2x
Embodiment
The expression of embodiment 1, Heparinase I fusion rotein MBP-HepA
1, the structure of expression vector pMal-hepA
The building process of expression vector pMal-hepA as shown in Figure 1, detailed process is as follows: amplification Heparinase I gene from the genome DNA of heparin Flavobacterium, used upstream and downstream primer is respectively 5 ' GCCT
GGATCCCAGCAAAAAAAATCCGGTAAC3 ' (base of band underscore is the restriction enzyme site of BamHI), 5 ' GCTT
CTGCAGTCTGGCAGTTTCGCTGTAC3 ' (base of band underscore is the PstI restriction enzyme site) introduces BamHI and PstI restriction enzyme site respectively, and the reaction system of amplification is: the 50ng template DNA, every kind of primer of 100pmol, 1 * amplification buffer, every kind of dNTP of 200 μ mol/L, the high Pfu enzyme of protecting of 1 unit; Amplification program is: 95 degrees centigrade of sex change 5 minutes, and 50-60 degree centigrade of primer annealing 45 seconds, 72 degrees centigrade of primer extensions 90 seconds, after 30 circulations, 72 degrees centigrade are extended and finished reaction in 5 minutes.This PCR result shows that amplification obtains the Heparinase I gene fragment of 1.1kb as shown in Figure 2.Among Fig. 2, it is 50,51,53,55,58 or 59 ℃ of amplifications that 1-6 is respectively the primer annealing temperature, and 7 is molecular weight marker 15kb, and arrow indication place is a 1.1kb target segment.
PMal-p2x and pMal-c2x carrier and PCR product are used BamHI and PstI double digestion respectively, connect with the T4DNA ligase enzyme, transform JM109, with 5 ' GCCTGGATCCCAGCAAAAAAAATCCGGTAAC3 ' and 5 ' GCTTCTGCAGTCTGGCAGTTTCGCTGTAC3 ' is primer, by bacterium colony PCR screening transformant, the upgrading grain is by BamHI and the checking of PstI double digestion again.Verify the result as shown in Figure 3, bacterium colony PCR result shows that about 55 ℃, the goal gene segment can access effective amplification.Transformed into escherichia coli TB1 obtains the recombinant plasmid vector bacterial strain that two strains contain correct connection, wherein a strain called after recombination bacillus coli TB1 (pMal-hepA).Among Fig. 3,1,8 is respectively 2k and λ/HindIII marker; 5-7 is the transformant of PCR checking, and 2-4 is 5-7 double digestion rear electrophoresis figure, and the arrow indication is a Heparinase I gene band; Wherein 6,7 is the correct pMal-hepA that connects.
2, the expression of Heparinase I fusion rotein MBP-HepA
Recombination bacillus coli TB1 (pMal-hepA) cultivated 3 hours for 37 ℃ at LB substratum (containing 100 μ g/ml Amp), added 1mM IPT6 and carried out inducing culture at 37 ℃ and 21 ℃ respectively.Get bacterium liquid at regular intervals and do full cell SDS-PAGE electrophoresis, induce back bacterium liquid to get 40ml centrifugal 10min under 10000rpm, with 20mM Tris-HCl washed twice, (output rating is 300W to be resuspended in ultrasonication among the 4ml 20mMTris.HCl, each ultrasonic 3 seconds and intermittently 3 seconds processing 99 times), get supernatant liquor 50 μ l and be SDS-PAGE and analyze.The result is shown in Fig. 4 a and Fig. 4 b, and different inducing temperatures are to the obvious effect of protein-active.Induce at 37 ℃, target protein exists with the form of non-activity inclusion body, and 21 ℃ induced, and the amount of soluble protein obviously increases.Among Fig. 4 a, 1,9 is protein marker, and 2-4 is an induction time 0,6,16 hours full cell electrophoresis figure, and 5,6 is 3,4 cytoclasis postprecipitation, and 7,8 is 3,4 supernatant liquor, and arrow indication place is fusion rotein MBP-hepA (94KDa).Among Fig. 4 b, 1-6 is induction time t=0,2,3,4,9,21 full cell electrophoresis figure, and 7 is molecular weight maerker; 8,9 is 5,6 cytoclasis postprecipitation, and 10,11 is 8,9 supernatant liquor, and arrow indication place is fusion rotein MBP-hepA (94KDa).
Recombination bacillus coli TB1 (pMal-hepA) cultivated 3 hours for 37 ℃ at LB substratum (containing 100 μ g/ml Amp), added 1mM IPTG respectively at 37 ℃, and 21 ℃ and 15 ℃ were carried out inducing culture 16 hours.After cell centrifugation separation, Tris buffer pH7.0 washed twice, be resuspended among the 4mlTris buffer, carry out cytoclasis.With broken liquid centrifugal after, the heparinase of surveying in the supernatant liquor is lived.Reddish black A (Azure A) method is adopted in the analysis that enzyme is lived, 30 ℃ of needed protein contents of 1mg substrate heparin of per hour degrading of definition that the enzyme of 1U is lived.The result shows that resulting heparinase work is respectively under the different inducing temperatures: 37 ℃, and 14U/L fermented liquid (2.9UL
-1OD
600 -1); 21 ℃, 89U/L fermented liquid (18.5UL
-1OD
600 -1), 15 ℃, 270U/L fermented liquid (108UL
-1OD
600 -1).
The fusion partners that utilizes among the present invention (fusion partner) maltose binding protein MBP can separate with affine one step of absorption realization of amylose starch.Concrete affine separating step is as follows: with 16 hours thalline 100ml of 0.3mM IPTG abduction delivering, the centrifugal 5min of 10000rpm; Establish the not thalline contrast of abduction delivering simultaneously.Then operate respectively by following two schemes:
Scheme one: (pH7.4) washed twice is resuspended among the 5ml column buffer for 20mM TrisHCl, 200mM NaCl, carries out ultrasonic (output rating is 300W, each ultrasonic 3 seconds and intermittently 3 seconds processing 99 times) with column equilibration liquid Column buffer.
Scheme two: osmotic pressure impacts.Thalline is resuspended in (20-40% sucrose, 30mMTrisHCl, 1mMEDTA) 15min, stirring among the 100ml osmotic shock buffer I.Centrifugal 10000rpm10min is resuspended in the equal-volume 0.5mM sal epsom ice bath 10-15min, centrifugal 10000rpm10min.
Centrifugal back supernatant is with the amylose starch affine separator column of 0.5ml/min by the 2ml pre-equilibration, by 10mM0.5ml/min maltose wash-out and collection.
Each step gets 50 μ l and is SDS-PAGE, after target protein process amylose starch (amylose) resin absorption, and can be under 1 column volume with 10mM maltose with the target protein wash-out.The result shows through target protein behind the amylose resin single step purification and can account for more than 95% as shown in Figure 5.Among Fig. 5,1 is the full cell electrophoresis of 0.3mM IPTG inductive E.coli TB1 (pMAL-hepA), 2 are the full cell electrophoresis of inductive E.coli TB1 (pMAL-hepA) not, 3 is molecular weight marker, and 4 is solvable target protein MBP-HEPA, and 5 is soluble target protein, 6-8 is through 1 of the maltose wash-out collection of amylose resin absorption back, 2,3 pipes (every pipe 1.5ml), the arrow indication is a target protein.
Sequence table
<160>2
<210>1
<211>2271
<212>DNA
＜213〉artificial sequence
<400>1
atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60
ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120
ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180
atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240
accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300
aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360
gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420
aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480
ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540
gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600
aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660
ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720
gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780
ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840
ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900
ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960
actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020
tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080
gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140
aacctcggga tcgagggaag gatttcagaa ttcggatccc agcaaaaaaa atccggtaac 1200
atcccttacc gggtaaatgt gcaggccgac agtgctaagc agaaggcgat tattgacaac 1260
aaatgggtgg cagtaggcat caataaacct tatgcattac aatatgacga taaactgcgc 1320
tttaatggaa aaccatccta tcgctttgag cttaaagccg aagacaattc gcttgaaggt 1380
tatgctgcag gagaaacaaa gggccgtaca gaattgtcgt acagctatgc aaccaccaat 1440
gattttaaga aatttccccc aagcgtatac caaaatgcgc aaaagctaaa aaccgtttat 1500
cattacggca aagggatttg tgaacagggg agctcccgca gctatacctt ttcagtgtac 1560
ataccctcct ccttccccga caatgcgact actatttttg cccaatggca tggtgcaccc 1620
agcagaacgc ttgtagctac accagaggga gaaattaaaa cactgagcat agaagagttt 1680
ttggccttat acgaccgcat gatcttcaaa aaaaatatcg cccatgataa agttgaaaaa 1740
aaagataagg acggaaaaat tacttatgta gccggaaagc caaatggctg gaaggtagaa 1800
caaggtggtt atcccacgct ggcctttggt ttttctaaag ggtattttta catcaaggca 1860
aactccgacc ggcagtggct taccgacaaa gccgaccgta acaatgccaa tcccgagaat 1920
agtgaagtaa tgaagcccta ttcctcggaa tacaaaactt caaccattgc ctataaaatg 1980
ccctttgccc agttccctaa agattgctgg attacttttg atgtcgccat agactggacg 2040
aaatatggaa aagaggccaa tacaattttg aaacccggta agctggatgt gatgatgact 2100
tataccaaga ataagaaacc acaaaaagcg catatcgtaa accagcagga aatcctgatc 2160
ggacgtaacg atgacgatgg ctattacttc aaatttggaa tttacagggt cggtaacagc 2220
acggtcccgg ttacttataa cctgagcggg tacagcgaaa ctgccagatg a 2271
<210>2
<211>756
<212>PRT
＜213〉artificial sequence
<400>2
Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys
1 5 10 15
Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30
Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe
35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala
50 55 60
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile
65 70 75 80
Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp
85 90 95
Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110
Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125
Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly
130 135 140
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160
Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys
165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly
180 185 190
Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205
Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220
Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240
Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser
245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro
260 265 270
Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285
Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala
290 295 300
Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala
305 310 315 320
Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350
Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn
355 360 365
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile
370 375 380
Glu Gly Arg Ile Ser Glu Phe Gly Ser Gln Gln Lys Lys Ser Gly Asn
385 390 395 400
Ile Pro Tyr Arg Val.Asn Val Gln Ala Asp Ser Ala Lys Gln Lys Ala
405 4l0 415
Ile Ile Asp Asn Lys Trp Val Ala Val Gly Ile Asn Lys Pro Tyr Ala
420 425 430
Leu Gln Tyr Asp Asp Lys Leu Arg Phe Asn Gly Lys Pro Ser Tyr Arg
435 440 445
Phe Glu Leu Lys Ala Glu Asp Asn Ser Leu Glu Gly Tyr Ala Ala Gly
450 455 460
Glu Thr Lys Gly Arg Thr Glu Leu Ser Tyr Ser Tyr Ala Thr Thr Asn
465 470 475 480
Asp Phe Lys Lys Phe Pro Pro Ser Val Tyr Gln Asn Ala Gln Lys Leu
485 490 495
Lys Thr Val Tyr His Tyr Gly Lys Gly Ile Cys Glu Gln Gly Ser Ser
500 505 510
Arg Ser Tyr Thr Phe Ser Val Tyr Ile Pro Ser Ser Phe Pro Asp Asn
515 520 525
Ala Thr Thr Ile Phe Ala Gln Trp His Gly Ala Pro Ser Arg Thr Leu
530 535 540
Val Ala Thr Pro Glu Gly Glu Ile Lys Thr Leu Ser Ile Glu Glu Phe
545 550 555 560
Leu Ala Leu Tyr Asp Arg Met Ile Phe Lys Lys Asn Ile Ala His Asp
565 570 575
Lys Val Glu Lys Lys Asp Lys Asp Gly Lys Ile Thr Tyr Val Ala Gly
580 585 590
Lys Pro Asn Gly Trp Lys Val Glu Gln Gly Gly Tyr Pro Thr Leu Ala
595 600 605
Phe Gly Phe Ser Lys Gly Tyr Phe Tyr Ile Lys Ala Asn Ser Asp Arg
610 615 620
Gln Trp Leu Thr Asp Lys Ala Asp Arg Asn Asn Ala Asn Pro Glu Asn
625 630 635 640
Ser Glu Val Met Lys Pro Tyr Ser Ser Glu Tyr Lys Thr Ser Thr Ile
645 650 655
Ala Tyr Lys Met Pro Phe Ala Gln Phe Pro Lys Asp Cys Trp Ile Thr
660 665 670
Phe Asp Val Ala Ile Asp Trp Thr Lys Tyr Gly Lys Glu Ala Asn Thr
675 680 685
Ile Leu Lys Pro Gly Lys Leu Asp Val Met Met Thr Tyr Thr Lys Asn
690 695 700
Lys Lys Pro Gln Lys Ala His Ile Val Asn Gln Gln Glu Ile Leu Ile
705 710 715 720
Gly Arg Asn Asp Asp Asp Gly Tyr Tyr Phe Lys Phe Gly Ile Tyr Arg
725 730 735
Val Gly Asn Ser Thr Val Pro Val Thr Tyr Asn Leu Ser Gly Tyr Ser
740 745 750
Glu Thr Ala Arg
755
Claims (12)
1, a kind of Heparinase I fusion rotein, its aminoacid sequence are as SEQ ID №: shown in 2.
2, Heparinase I fusion rotein encoding gene is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.
3, gene according to claim 2 is characterized in that: described Heparinase I fusion rotein encoding gene is the SEQ ID № in the sequence table: 1.
4, contain claim 2 or 3 described expression carrier.
5, expression vector according to claim 4 is characterized in that: described expression vector is pMAL-hepA.
6, the transgenic cell line that contains claim 2 or 3 described genes.
7, the engineering bacteria that contains claim 2 or 3 described genes.
8, engineering bacteria according to claim 7 is characterized in that: described engineering bacteria is the intestinal bacteria that contain described Heparinase I fusion rotein encoding gene.
9, a kind of method of expressing the Heparinase I fusion rotein is that the recombinant expression vector that will contain Heparinase I fusion rotein encoding gene imports the expressive host bacterium, expresses obtaining expressing the Heparinase I fusion rotein; Described Heparinase I fusion rotein encoding gene is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.
10, method according to claim 9 is characterized in that: the recombinant expression vector of described Heparinase I fusion rotein encoding gene is pMAL-hepA; Described host bacterium is intestinal bacteria.
11, according to claim 9 or 10 described methods, it is characterized in that: the abduction delivering condition of described Heparinase I fusion rotein is 0.01-1mM IPTG, 10-42 degree centigrade and induced 16 hours; Yeast extract concentration in the substratum that is adopted is 1g/L～20g/L; Also add 1%-5% ethanol and/or 0.05-2mg/L kantlex and/or 0.01-2.0mg/L paraxin in the described substratum.
12, method according to claim 11 is characterized in that: described expression condition is that engineering bacteria, adds 0.3mM IPTG and induced 16 hours at 15 degrees centigrade after 3 hours 37 degrees centigrade of cultivations; The substratum that is adopted contains NaCl 10g/L, yeast extract paste 7.5g/L, peptone 10g/L, 1% ethanol, 0.6mg/L paraxin.
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| US7691613B2 (en) | 2006-11-03 | 2010-04-06 | Momenta Pharmaceuticals, Inc. | Glycosaminoglycan lyase IV and uses thereof |
| US7691612B2 (en) | 2005-11-03 | 2010-04-06 | Momenta Pharmaceuticals, Inc. | Heparan sulfate glycosaminoglycan lyase and uses thereof |
| US7767420B2 (en) | 2005-11-03 | 2010-08-03 | Momenta Pharmaceuticals, Inc. | Heparan sulfate glycosaminoglycan lyase and uses thereof |
| CN101294177B (en) * | 2008-05-26 | 2012-07-18 | 清华大学 | Method for preparing low molecular weight heparin |
| CN101608178B (en) * | 2009-07-29 | 2011-05-18 | 清华大学 | Fusion heparinase and coding gene and preparation method thereof |
| CN101608180B (en) * | 2009-07-29 | 2011-08-10 | 清华大学 | Fusion heparinase and coding gene thereof |
| CN101942025B (en) * | 2010-08-20 | 2012-07-04 | 清华大学 | Heparanase III fusion protein and coding gene and expression method thereof |
| CN101942024B (en) * | 2010-08-20 | 2012-07-04 | 清华大学 | Heparanase II fusion protein and coding gene and expression method thereof |
| CN104073480A (en) * | 2014-07-04 | 2014-10-01 | 深圳市海普瑞药业股份有限公司 | High-immobilization-tendency heparinase I coding gene and protein thereof |
| 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 |
| US10900028B2 (en) | 2016-12-22 | 2021-01-26 | Shenzhen Hepalink Pharmaceutical Group Co., Ltd. | Heparinase-producing Pseudomonas stutzeri strain and heparinase derived therefrom |
| CN114181927B (en) * | 2021-08-18 | 2023-08-18 | 刘颖 | Heparinase I |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997011684A1 (en) * | 1995-09-29 | 1997-04-03 | Ibex Technologies Inc. | Use of heparinases to decrease inflammatory responses |
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2004
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997011684A1 (en) * | 1995-09-29 | 1997-04-03 | Ibex Technologies Inc. | Use of heparinases to decrease inflammatory responses |
Non-Patent Citations (4)
| Title |
|---|
| Immobilization of recombinant heparinase I fused tocellulose-binding domain Shpigel.E et al,Biotechnol.Bioeng.,Vol.65 No.1 1999 * |
| Immobilization of recombinant heparinase I fused tocellulose-binding domain Shpigel.E et al,Biotechnol.Bioeng.,Vol.65 No.1 1999;肝素枰产生菌的筛选及发酵条件 高宁国等，微生物学报，第39卷第1期 1999;一种改进的融合蛋白亲和层析纯化方法 廉德君 许根俊，生物化学与生物物理进展，第25卷第3期 1998 * |
| 一种改进的融合蛋白亲和层析纯化方法 廉德君 许根俊，生物化学与生物物理进展，第25卷第3期 1998 * |
| 肝素枰产生菌的筛选及发酵条件 高宁国等，微生物学报，第39卷第1期 1999 * |
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Application publication date: 20051123 Assignee: BEIJING BICHENG BIOTECHNOLOGY Co.,Ltd. Assignor: Tsinghua University Contract record no.: 2015990000925 Denomination of invention: Heparinase I fusion protein and genes encoding same and expression method thereof Granted publication date: 20070425 License type: Common License Record date: 20151109 |
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