CN1766107A - Recombinant human keratinized cell growth factor production method - Google Patents
Recombinant human keratinized cell growth factor production method Download PDFInfo
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- CN1766107A CN1766107A CN 200510085496 CN200510085496A CN1766107A CN 1766107 A CN1766107 A CN 1766107A CN 200510085496 CN200510085496 CN 200510085496 CN 200510085496 A CN200510085496 A CN 200510085496A CN 1766107 A CN1766107 A CN 1766107A
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- lys
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Abstract
The invention relates to a preparation method for recombinant human horny cell growth factor, special to prepare biological active polypeptide, which comprises: a. synthesizing artificially the gene of amino acid coding region of objective product; b. fusing the obtained gene with proper 3' end of S- transferase protein gene of GSH on expression carrier contained thrombin or enterokinase recognition site; c. conversing obtained expression carrier into proper escherichia coli to obtain genetic engineering fungus; d. fermenting the fungus, extracting, chromatography, purifying, and obtaining the GST-KGF fused protein; e. cutting by thrombin or enterokinase, separating and purifying, and obtaining the objective factor.
Description
Technical field:
The present invention relates to a kind of preparation method of biologically active polypeptides, particularly a kind of method of utilizing gene recombination technology to prepare the biologically active polypeptides of human keratinocyte Growth Factor.
Background technology:
Human keratinocyte Growth Factor (English name: Human Keratinocyte Growth factor, be called for short: KGF or KGF1) is knownly can impel non-one-tenth fiber epithelial cell (particularly keratinocyte) the proliferating cells factor at present.The histology of KGFmRNA is distributed with its singularity: the expression of KGFmRNA is arranged in several matrix fibroblasts from embryo, newborn infant and adult's epithelium, expression is also arranged in people's kidney, gi tract, and in spongiocyte, lung, brain and various epithelial cell (comprising squamous cell carcinoma, bronchial epithelial cell etc.), do not detect KGF mRNA.This histology of KGF mRNA distributes that prompting KGF produces and not subcutaneous in corium.KGF has the mitogenesis effect to mouse BALB/MK cell, and KGF has differentiation and the maturation that influences keratinocyte simultaneously.(Rubin et al.Proc.Natl.Acad.Sci.USA，86：802-806(1989))。Simultaneously, KGF also has hormesis to the non-keratinocyte epithelial cell: in vivo, KGF induces normal skin accessory structure (as cortex cell and hair follicle cell), the hyperplasia and the differentiation of liver cell and respiratory film epithelial cell (as II type pneumonocyte), stimulate the hyperplasia and the growth of intestinal mucosa cell (producing mucoprotein goblet cell as the gastric gland intestinal glands, other epithelial cell in the pit cell of colon and the enteron aisle).(WO94/23032)。
At present KGF has been used for the treatment of the digestive tract ulcer that the blood of human body tumor chemoradiotherapy causes.
Preparation KGF mainly uses recombinant expression method at present, in W90/108771, WO96/11951 description is arranged.
The prior art processes step is extremely numerous and diverse, simultaneously, KGF has extremely strong toxicity to escherichia coli host, and KGF is very low in common expression in escherichia coli efficient, for overcoming above defective, this patent provides a kind of preparation method who utilizes the method preparation reorganization KGF of amalgamation and expression.This method has been simplified technology greatly, has improved the output of KGF greatly, thus but large-scale industrial production KGF.
Summary of the invention:
The invention provides a kind of method for preparing human keratinocyte Growth Factor, this method may further comprise the steps:
A. the corresponding gene in synthetic human keratinocyte Growth Factor amino acid coding region;
B. glutathione S-transferase (the GlutathioneS-transferase:GST) protein gene 3 ' end on step a gene that obtains and the expression vector that suits merges, and this gene middle portion contains zymoplasm or enteropeptidase recognition site;
The suitable expression vector that c. will contain the fusion rotein encoding gene transforms suitable intestinal bacteria and obtains genetic engineering bacterium;
D. this genetic engineering bacterium through extracting the chromatography purification step, is prepared the GST-KGF fusion rotein after fermentation;
E. again after the cutting of zymoplasm or enteropeptidase, separation and purification obtains recombinant human horny cell growth factor-2.
The corresponding gene in wherein said synthetic human keratinocyte Growth Factor amino acid coding region is a prior art, can obtain by prior art, as the method for synthetic.
Glutathione S-transferase described in the step b. (Glutathione S-transferase:GST) protein gene is a prior art, can on market, buy and obtain, cut recognition site-(ASP) N Lys-connection by the enteropeptidase enzyme between GST albumen wherein and the human keratinocyte Growth Factor, structure is X__ (ASP) N Lys_KGF1, wherein (Asp) is aspartic acid, X is GST, and n is 2,3 or 4.Or
Connect by zymoplasm recognition site X-P2-P3-Pro-P1. between GST albumen and the human keratinocyte Growth Factor in the described fusion rotein, mode of connection is X-P2-P3-Pro-P1_KGF1, wherein X is GST, P1 is Arg or Lys, P2, P3 are hydrophobic amino acid (being selected from: Leu, Val, ILe, Met, Ala, Cys or Trp), the preferred Leu of P2 here, the preferred Val of P3, its aminoacid sequence is preferably X-Leu-Val-Pro-Arg-KGF1, and wherein X is GST.
The DNA sequences encoding of the preferred GST-KGF1 fusion rotein of the present invention is seen sequence table 1 and 3.
The aminoacid sequence of GST-KGF1 fusion rotein of the present invention is seen sequence table 2 and 4.
Preparation method of the present invention is characterized in that, wherein said fermentation is qualified recombinant human horny cell growth factor-2 genetic engineering bacterium to be inoculated in the fermentor tank cultivate, and wherein adds IPTG and induces Expression of Fusion Protein.
Preparation method of the present invention, wherein said extraction chromatography purification step (1) comprises centrifugal collection thalline, broken bacterium, the step of the broken bacterium liquid supernatant of centrifugal collection, with Heparin affinity column on the supernatant of collecting, with level pad flush away foreign protein, then with the NaCl wash-out GST-KGF1 fusion rotein that contains suitable concentration, with Sepharose 6B affinity column on the GST-KGF1 fusion rotein of wash-out, GSH wash-out fusion rotein with proper concn, the gained fusion rotein is done enzyme with highly purified Enterkinase or Thrombin and is cut processing, SP Sepharose F.F. post removes GST albumen on the fusion rotein that enzyme is cut, the KGF1 that obtains is gone up Heparin affinity chromatography column chromatography, with the NaCl wash-out KGF1 that contains suitable concentration, obtain the pure KGF1 of purity more than 95%.
The present invention also provides the pharmaceutical composition with the human keratinocyte Growth Factor of method of the present invention preparation, and said composition exists with pharmaceutical dosage forms, freeze dried injection preferably, and the injection of unitary dose is every injection, its prescription is composed as follows:
Keratinocyte growth factor 6.6mg
N.F,USP MANNITOL 5～20mg
Citric acid (water) 2.1mg
Lemon sodium (two water) 2.94mg or
Keratinocyte growth factor 6.6mg
N.F,USP MANNITOL 5～20mg
SODIUM PHOSPHATE, MONOBASIC 0.93mg
Sodium phosphate dibasic 1.73mg
Sodium-chlor 10mg
The present invention is according to the amino acid coding of the human keratinocyte Growth Factor primary structure of having delivered (this sequence can change the preference principle that genetic code uses according to versatility, degeneracy and the intestinal bacteria of genetic code), this double-stranded DNA complete sequence of synthetic.Principal feature of the present invention is, respectively this encoding gene fragment cloning is gone into suitable expression vector, (preferred as: pGEX-4T-3, can buy from the market) the middle formation recombinant plasmid that makes up, then that recombinant plasmid transformed is suitable intestinal bacteria, preferred as: BL21 (DE3) (can buy from the market), the genetic engineering bacterium that obtains through screening with high expression level ability.This genetic engineering bacterium is fusion partner with GST, amalgamation and expression KGF1, promptly form the GST-KGF1 fusion rotein, after the separation and purification of expressed fusion protein process suitable step, with suitable proteolytic enzyme with distinguished sequence recognition capability as KGF1 being cut down from fusion rotein with enteropeptidase (Enterkinase) or zymoplasm (Thrombin), through the high separation purifying, obtain the pure product of KGF1.Through analyses such as-terminal amino acid sequencing, mass spectrum molecular weight determination, isoelectric point determination, peptide figure analysis and biological activity assay, the result shows that human keratinocyte Growth Factor that we are expressed and natural each index of human keratinocyte Growth Factor are in full accord.
The ultimate principle of the molecular cloning method that the present invention uses, and employed raw material in the operation steps, solvent, instrument belongs to the prior art field, as the synthetic method of DNA, and the selection of restriction enzyme site etc., the expression vector that the present invention uses, host bacterium etc. all is commercialization carrier or bacterial classification.
The concrete operations step of above method can be seen embodiments of the invention.
Advantage of the present invention mainly shows:
(1), there be (connecting with enteropeptidase Enterkinase recognition sequence) in the GST-KGF1 fusion rotein with soluble form among the present invention, thus simplify production technique greatly and enhance productivity greatly;
(2), the KGF1 that obtains by above-mentioned technology, the structure of physicochemical properties such as its structure and biologic activity and bibliographical information is in full accord, avoid common recombinant protein drug N end front to have more a Met even a plurality of amino-acid residue, thereby can reduce its immunogenicity;
Description of drawings:
Fig. 1 pGEX-4T-3 plasmid map and multiple clone site and sequence
Fig. 2 pGEX-4T-3-KGF1 construction of recombinant plasmid synoptic diagram
Fig. 3 GST-E-KGF1 fusion rotein (enteropeptidase recognition sequence) expressing fusion protein, enteropeptidase are handled the human keratinocyte Growth Factor electrophoretogram of back purifying
Embodiment:
Further specify the present invention by the following examples, but not as limitation of the present invention.
1. the selection of expression vector
Select for use the pGEX series expression vector of Novagen company such as pGEX-4T-3 as expression vector, pGEX-4T-3 is the coli expression carrier system that is used for efficient amalgamation and expression albumen and polypeptide, target protein or polypeptide with the glutathione s-transferase (GST) of 225 amino-acid residues as fusion partner.(seeing accompanying drawing 1).
2. the design of gene and synthetic:
(sequence can change according to versatility, the degeneracy principle of genetic code according to the KGF1 dna encoding sequence of bibliographical information, but not changing that amino acid is formed and arrange) design is encoded into acquaintance KGF1 sequence, adds BamHI recognition sequence GGATCC and enteropeptidase (Enterokinase) restriction enzyme site encoding sequence or zymoplasm (thrombin) restriction enzyme site encoding sequence at 5 ' of this sequence-end simultaneously; Add the recognition sequence CTCGAG of Xho I at 3 ' end;
3. the nucleic acid fragment that above-mentioned synthetic is had different restriction enzyme sites is inserted into the Kpn of pGEX-4T-3 and Xho I site is formed sequence 2 or sequence 4 jointly by the KGF1 dna encoding sequence of proteic encoding sequence of fusion partner GST on the expression vector and insertion NDA sequence respectively, the aminoacid sequence of this sequence encoding is sequence 1 and sequence 3, sees sequence table 1,2,3,4.Form recombinant plasmid, transformed into escherichia coli BL21 efficiently expresses genetic engineering bacterium through screening promptly to get.Relevant working method is referring to " molecular cloning handbook cold spring port second edition; Make up collection of illustrative plates and see accompanying drawing 2
With microbionation in LB or other suitable culture base, long during to suitable concentration such as mid-log phase when engineering bacteria, add suitable concentration IPTG (as 0.1～0.5mM), visible significantly expressing fusion protein band;
5. with the seed liquor of overnight incubation, be inoculated in the fermentor tank suitable culture base (in LB, TB or other appropriate media) and cultivate, microbial culture adds IPTG inducing culture certain hour (as: 3-4 hour) during to suitable degree.Centrifugal receipts bacterium is suspended in (as phosphate buffered saline buffer, Tris-HCL damping fluid etc.) in the suitable damping fluid with thalline, the broken bacterium of excusing from death ripple or high-pressure homogenization.The bacterium liquid of fragmentation is centrifugal, collect supernatant liquor, abandon precipitation.
With Heparin affinity column on the supernatant of collecting, with level pad flush away foreign protein, then with the NaCl wash-out GST-KGF1 fusion rotein that contains suitable concentration, with Sepharose 6B affinity column on the GST-KGF1 fusion rotein of wash-out, GSH wash-out fusion rotein with proper concn, the gained fusion rotein is done enzyme with highly purified Enterkinase or Thrombin and is cut processing, SP Sepharose F.F. post removes GST albumen on the fusion rotein that enzyme is cut, the KGF1 that obtains is gone up Heparin affinity chromatography column chromatography, with the NaCl wash-out KGF1 that contains suitable concentration, obtain the pure KGF1 of purity more than 95%.The KGF1 electrophoresis accompanying drawing 3 that obtains behind the purifying:
Embodiment 2
Physical and chemical property determining according to the rhKGF of embodiment 1 gained
1, terminal 15 determined amino acid sequences of N-
With above-mentioned two kinds of recombinant human horny cell growth factor-2s that method obtains, carry out the terminal mensuration of N-, measurement result is as follows:
N-Ser-Tyr-Asp-Tyr-Met-Glu-Gly-Gly-Asp-Ile-Arg-Val-Arg-Ar g-Leu-C and expected results are in full accord.
2, amount mass spectroscopy
Adopt the FAB method to measure, the molecular weight of the recombinant human horny cell growth factor-2 that two kinds of methods obtain is, 16278.5, and consistent with theoretical prediction.
3, Determination of biological activity:
Utilize KGF1 to promote the characteristics of 4MBr-5 cell proliferation to carry out the bioactive mensuration of KGF1.Recording the result is:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | |
| Standard substance | 0.185 | 0.182 | 0.138 | 0.131 | 0.109 | 0.111 | 0.102 | 0.103 | 0.105 | 0.094 | 0.105 |
| 0.194 | 0.155 | 0.15 | 0.117 | 0.114 | 0.104 | 0.116 | 0.108 | 0.095 | 0.108 | 0.091 | |
| 0.184 | 0.168 | 0.14 | 0.134 | 0.127 | 0.115 | 0.108 | 0.111 | 0.106 | 0.102 | 0.097 | |
| Mean value | 0.188 | 0.168 | 0.145 | 0.127 | 0.117 | 0.11 | 0.109 | 0.107 | 0.102 | 0.101 | 0.098 |
| Product to be tested | 0.195 | 0.169 | 0.15 | 0.13 | 0.116 | 0.108 | 0.106 | 0.107 | 0.098 | 0.114 | 0.094 |
| 0.19 | 0.17 | 0.153 | 0.126 | 0.125 | 0.118 | 0.12 | 0.102 | 0.11 | 0.096 | 0.09 | |
| 0.187 | 0.18 | 0.146 | 0.137 | 0.108 | 0.11 | 0.101 | 0.108 | 0.102 | 0.09 | 0.114 | |
| Mean value | 0.191 | 0.173 | 0.15 | 0.131 | 0.116 | 0.112 | 0.109 | 0.106 | 0.103 | 0.1 | 0.099 |
| Negative | 0.107 | 0.094 | 0.1 | 0.095 |
Sequence table
＜110〉Chengdu Zhitian Bioengineering Co., Ltd.
＜120〉a kind of production method of recombinant human horny cell growth factor-2
<160>4
<210>1
<211>1101
<212>DNA
＜213〉artificial sequence
<400>1
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gttcctacga ctacatggaa ggtggtgaca tccgtgttcg tcgtctgttc 720
tgccgtaccc agtggtacct gcgtatcgac aaacgtggta aagttaaagg tacccaggaa 780
atgaaaaaca actacaacat catggaaatc cgtaccgttg ctgttggtat cgttgctatc 840
aaaggtgttg aatccgaatt ctacctggct atgaacaaag aaggtaaact gtacgctaaa 900
aaagaatgca acgaagactg caacttcaaa gaactgatcc tggaaaacca ctacaacacc 960
tacgcttccg ctaaatggac ccacaacggt ggtgaaatgt tcgttgctct gaaccagaaa 1020
ggtatcccgg ttcgtggtaa aaaaaccaaa aaagaacaga aaaccgctca cttcctgccg 1080
atggctatca cctaactcgag 1101
<210>2
<211>364
<212>PRT
＜213〉artificial sequence
<400>2
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln
5 10 15
Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu
20 25 30
His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys
35 40 45
Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp
50 55 60
Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile
65 70 75
Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
80 85 90
Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly
95 100 105
Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val
110 115 120
Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp
125 130 135
Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
140 145 150
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met
155 160 165
Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
170 175 180
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser
185 190 195
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe
200 205 210
Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Ser
215 220 225
Tyr Asp Tyr Met Glu Gly Gly Asp Ile Arg Val Arg Arg Leu Phe
230 235 240
Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp Lys Arg Gly Lys Val
245 250 255
Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn Ile Met Glu Ile
260 265 270
Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly Val Glu Ser
275 280 285
Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr Ala Lys
290 295 300
Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu Glu
305 310 315
Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly
320 325 330
Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg
335 340 345
Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro
350 355 360
Met Ala Ile Thr
364
<210>3
<211>1122
<212>DNA
＜213〉artificial sequence
<400>3
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatccga tgacgatgac aagtcctacg actacatgga aggtggtgac 720
atccgtgttc gtcgtctgtt ctgccgtacc cagtggtacc tgcgtatcga caaacgtggt 780
aaagttaaag gtacccagga aatgaaaaac aactacaaca tcatggaaat ccgtaccgtt 840
gctgttggta tcgttgctat caaaggtgtt gaatccgaat tctacctggc tatgaacaaa 900
gaaggtaaac tgtacgctaa aaaagaatgc aacgaagact gcaacttcaa agaactgatc 960
ctggaaaacc actacaacac ctacgcttcc gctaaatgga cccacaacgg tggtgaaatg 1020
ttcgttgctc tgaaccagaa aggtatcccg gttcgtggta aaaaaaccaa aaaagaacag 1080
aaaaccgctc acttcctgcc gatggctatc acctaactcg ag 1122
<210>4
<211>371
<212>PRT
＜213〉artificial sequence
<400>4
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln
1 5 10 15
Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu
20 25 30
His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys
35 40 45
Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp
50 55 60
Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile
65 70 75
Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala
80 85 90
Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly
95 100 105
Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val
110 115 120
Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp
125 130 135
Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
140 145 150
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met
155 160 165
Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
170 175 180
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser
185 190 195
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe
200 205 210
Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly
215 220 225
Ser Asp Asp Asp Asp Lys Ser Tyr Asp Tyr Met Glu Gly Gly Asp
230 235 240
Ile Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg
245 250 255
Ile Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn
260 265 270
Asn Tyr Asn Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val
275 280 285
Ala Ile Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys
290 295 300
Glu Gly Lys Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn
305 310 315
Phe Lys Glu Leu Ile Leu Glu Asn His Tyr Asn Thr Tyr Ala Ser
320 325 330
Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val Ala Leu Asn
335 340 345
Gln Lys Gly Ile Pro Val Arg Gly Lys Lys Thr Lys Lys Glu Gln
350 355 360
Lys Thr Ala His Phe Leu Pro Met Ala Ile Thr
365 370 371
Claims (10)
1, a kind of preparation method of human keratinocyte Growth Factor may further comprise the steps:
A. the corresponding gene in synthetic human keratinocyte Growth Factor amino acid coding region;
B. the GST protein gene 3 ' end on step a gene that obtains and the expression vector that suits merges, and the centre contains enteropeptidase or zymoplasm recognition site;
The suitable expression vector that c. will contain the fusion rotein encoding gene transforms suitable intestinal bacteria and obtains genetic engineering bacterium;
D. this genetic engineering bacterium through extracting the chromatography purification step, is prepared the GST-KGF fusion rotein after fermentation;
E. again after the cutting of enteropeptidase or zymoplasm, separation and purification obtains recombinant human horny cell growth factor-2.
2, preparation method according to claim 1 is characterized in that, cuts recognition site-(Asp) by the enteropeptidase enzyme between GST albumen and the human keratinocyte Growth Factor in the above-mentioned fusion rotein
N-Lys-connects, and structure is X-(Asp)
N-Lys-KGF, wherein Asp is an aspartic acid, and X is a GST albumen, and n is 2,3 or 4.
3, preparation method according to claim 1, it is characterized in that, cutting recognition site X-P2-P3-Pro-P1. by zymoplasm (Thrombin) enzyme between GST albumen and the human keratinocyte Growth Factor in the wherein said fusion rotein connects, mode of connection is X-P4-P3-Pro-P1-KGF, wherein X is a GST albumen, P1 is Arg or Lys, and P2, P3 are hydrophobic amino acid.
4, preparation method according to claim 1 is characterized in that, wherein said hydrophobic amino acid is selected from Leu, Val, ILe, Met, Ala, Cys or Trp.
5, preparation method according to claim 3, it is characterized in that, among the structural formula X-P4-P3-Pro-P1-KGF, P1 is Arg, P2 is that Val, P3 are Leu, its sequence expression formula is X-Leu-Val-Pro-Arg-KGF, and wherein X is a GST albumen, and the KGF sequence lacks 140 the amino acid whose KGF that contain of 23 amino-acid residues of N-end for the people who has reported.
6. preparation method according to claim 1 is characterized in that the DNA sequences encoding of wherein said GST-KGF fusion rotein is seen sequence table 1 and 3.
7. preparation method according to claim 1 is characterized in that the aminoacid sequence of wherein said GST-KGF fusion rotein is seen sequence table 2 and 4.
8. preparation method according to claim 2 is characterized in that, wherein said fermentation is qualified recombinant human horny cell growth factor-2 genetic engineering bacterium to be inoculated in the fermentor tank cultivate, and wherein adds IPTG and induces Expression of Fusion Protein.Extract the chromatography purification step and comprise centrifugal collection thalline, broken bacterium, the step of the broken bacterium liquid supernatant of centrifugal collection, with Heparin affinity column on the supernatant of collecting, with level pad flush away foreign protein, then with the NaCl wash-out GST-KGF fusion rotein that contains suitable concentration, with Sepharose 6B affinity column on the GST-KGF fusion rotein of wash-out, GSH wash-out fusion rotein with proper concn, the gained fusion rotein is done enzyme with highly purified Enterkinase or Thrombin and is cut processing, SP Sepharose F.F. post removes GST albumen on the fusion rotein that enzyme is cut, the KGF that obtains is gone up Heparin affinity chromatography column chromatography, with the NaCl wash-out KGF that contains suitable concentration, obtain the pure KGF of purity more than 95%.
9, the pharmaceutical composition of the human keratinocyte Growth Factor of the method preparation of usefulness claim 1, its prescription is composed as follows:
Human keratinocyte Growth Factor 0.5mg
N.F,USP MANNITOL 5～20mg
Citric acid (water) 2.1mg
Lemon sodium (two water) 2.94mg
10, the pharmaceutical composition of the human keratinocyte Growth Factor of the method preparation of usefulness claim 1, its prescription is composed as follows:
Human keratinocyte Growth Factor 0.5mg
N.F,USP MANNITOL 5～20mg
SODIUM PHOSPHATE, MONOBASIC 0.93mg
Sodium phosphate dibasic 1.73mg
Sodium-chlor 10mg
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242124A (en) * | 2010-05-10 | 2011-11-16 | 齐鲁制药有限公司 | Modified Keratinocyte growth factor gene and its expression in yeast |
| CN103952427A (en) * | 2014-05-11 | 2014-07-30 | 浙江大学 | Human papillomavirus (HPV) 6bE7 protein expression method and application |
| CN104059933A (en) * | 2014-05-11 | 2014-09-24 | 浙江大学 | Expression and application of human papilloma virus 11E7 protein |
| CN101721683B (en) * | 2009-11-10 | 2015-06-24 | 苏州金盟生物技术有限公司 | Application of human Keratiocyte growth factor 1 in preparation of medicament for treating anal fissure |
| CN106226511A (en) * | 2016-07-28 | 2016-12-14 | 苏州金盟生物技术有限公司 | A kind of recombinant human horny cell growth factor-2 biologic activity detection method |
| CN109456399A (en) * | 2017-09-06 | 2019-03-12 | 山东益华生物科技有限公司 | Application of the plant as host in expression keratinocyte growth factor |
| CN113474361A (en) * | 2019-02-24 | 2021-10-01 | 昂科斯密斯生物技术私人有限公司 | Method for continuous production of recombinant GLP-1 peptides from bacteria |
-
2005
- 2005-07-22 CN CNB2005100854968A patent/CN100545264C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721683B (en) * | 2009-11-10 | 2015-06-24 | 苏州金盟生物技术有限公司 | Application of human Keratiocyte growth factor 1 in preparation of medicament for treating anal fissure |
| CN102242124A (en) * | 2010-05-10 | 2011-11-16 | 齐鲁制药有限公司 | Modified Keratinocyte growth factor gene and its expression in yeast |
| CN102242124B (en) * | 2010-05-10 | 2013-05-22 | 齐鲁制药有限公司 | Modified Keratinocyte growth factor gene and its expression in yeast |
| CN103952427A (en) * | 2014-05-11 | 2014-07-30 | 浙江大学 | Human papillomavirus (HPV) 6bE7 protein expression method and application |
| CN104059933A (en) * | 2014-05-11 | 2014-09-24 | 浙江大学 | Expression and application of human papilloma virus 11E7 protein |
| CN106226511A (en) * | 2016-07-28 | 2016-12-14 | 苏州金盟生物技术有限公司 | A kind of recombinant human horny cell growth factor-2 biologic activity detection method |
| CN106226511B (en) * | 2016-07-28 | 2018-04-06 | 苏州金盟生物技术有限公司 | A kind of recombinant human horny cell growth factor-2 biological activity detection method |
| CN109456399A (en) * | 2017-09-06 | 2019-03-12 | 山东益华生物科技有限公司 | Application of the plant as host in expression keratinocyte growth factor |
| CN113474361A (en) * | 2019-02-24 | 2021-10-01 | 昂科斯密斯生物技术私人有限公司 | Method for continuous production of recombinant GLP-1 peptides from bacteria |
| CN113474361B (en) * | 2019-02-24 | 2023-11-14 | 昂科斯密斯生物技术私人有限公司 | Method for continuous production of recombinant GLP-1 peptide by bacteria |
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| CN100545264C (en) | 2009-09-30 |
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