CN1888066A - Production process of recombinant human horny cell growth factor-2 - Google Patents

Production process of recombinant human horny cell growth factor-2 Download PDF

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CN1888066A
CN1888066A CN 200510027206 CN200510027206A CN1888066A CN 1888066 A CN1888066 A CN 1888066A CN 200510027206 CN200510027206 CN 200510027206 CN 200510027206 A CN200510027206 A CN 200510027206A CN 1888066 A CN1888066 A CN 1888066A
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rhkgf
growth factor
expression vector
cell growth
chromatography
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CN100487118C (en
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任军
黄阳滨
孙九如
丰涛
杜碧金
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Zhejiang Thirty Thousand Pharmaceutical Co ltd
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Shanghai Newsummit Biopharma Co Ltd
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Abstract

The present invention is the coding sequence and production process of optimized human horny cell growth factor-2 (rhKGF-2), and expression vector and host cell for the production process. The optimized rhKGF-2 series of the present invention has high expression amount and simple separating and purifying process. The present invention also provides corresponding expression vector and engineering bacterium.

Description

A kind of production method of recombinant human keratinized cell growth factor-2
Technical field
The present invention relates to the genetically engineered field, relate more specifically to a kind of human body keratinized cell growth factor-2 (rhKGF-2) encoding sequence of optimization, the method for producing recombinant human keratinized cell growth factor-2, and the expression vector and the host cell that are used for this method.
Background technology
(Recombinant Human Keratinocyte GrowthFactor-2 is that country's treatment of intractables such as the mucositis that causes of treatment diabetic venous ulcer, ulcerative colitis, high dose chemotherapy, burn and scald, chronic trauma is with biological products a kind new medicine rhKGF-2) to recombinant human keratinized cell growth factor-2.It can stimulate epithelial propagation specifically, impels epidermal growth and granulation tissue to form simultaneously, and the unnecessary collagen of degrading reduces the generation of scar.
RhKGF-2 is listed in the FGF family member by sequence homology analysis at first, be called FGF-10 again, this family is extended familys that participate in soft tissue growth, 18 members of Fa Xianing all have wide mitogenesis spectrum so far, and promptly they can promote the propagation and/or the promotion vascularization of the various cells of mesoderm and neuroectodermal origin.RhKGF-2 is made up of 208 amino acid, it can be incorporated into fibroblast growth factor acceptor 2 IIIb (the fibroblast growth factor recptor-2IIIb on target cell surface specifically, FGFR2IIIb) and fibroblast growth factor acceptor 1IIIb (fibroblast growth factor recptor-1IIIb, FGFR1IIIb), two acceptor triggering signal conduction, thereby performance physiological action.Because FGFR mainly is present in the epithelium, therefore rhKGF-2 can stimulate epithelial propagation specifically, impel epidermal growth and granulation tissue to form simultaneously, as stimulating liver, kidney, pancreas, the propagation of Urothelial Cell and the increase of gastrointestinal tract cell quantity; It is by activating collagenase simultaneously, too much collagen in the degraded scar tissue, the hyperplasia that suppresses the pathologic scar, do not stay obvious scar after cicatrizing a wound, it is a kind of protein factor that wound healing is had very much the treatment potentiality, therefore all have the injury region of epithelium all can use this product, so play an important role aspect wound healing.
Repifermin is U.S. HUMAN GENOME SCIENCES, the rhKGF-2 product of Inc (HGS) exploitation.Begin in February, 1998 to carry out two I phase experimental studies that rhKGF-2 is used for wound healing, one of them is the local application of skin surface, and another is the systemic administration by injection.The purpose of test is to estimate security, pharmacokinetics and the appropriate dosage of rhKGF-2.Test-results shows that under selected dosage, it is safe using rhKGF-2 in the body system, and the healthy volunteer can be well tolerable, almost has no side effect.April 5 calendar year 2001, HGS company began more massive IIb test, investigated the effectiveness that venous ulcer fully healed of 700 patients with further evaluation KGF-2 at 70 clinical trial points of the U.S..On April 2nd, 2003, the Repifermin that is through with of HGS company studied at the clinical II of the mucositis phase that chemotherapy causes, the result shows that the Repifermin security is good, and evident in efficacy.
Present international rhKGF-2 escherichia expression system production technique, no matter whether its expression product exist with the inclusion body form, because of codon without optimization, carrier is selected inappropriate, cause expression amount low, and must could obtain the result that purity is 90% finished product through 4-5 step chromatography purification method.
A kind of method of utilizing escherichia expression system to produce human body keratinized cell growth factor-2 is disclosed in Chinese patent application CN02110808.0.Though this method is optimized the expression vector of selecting for use, make expression amount be improved (about 200ug/ml), and optimized separation purifying technique, yet because the hKGF-2 encoding sequence of selecting for use remains natural people hKGF-2 sequence, so the raising of expression amount still is very limited.
Yet the efficient with intestinal bacteria production human body keratinized cell growth factor-2 is also lower so far.Therefore, this area presses for the technology of the production human body keratinized cell growth factor-2 of the new high-efficient simple of exploitation.
Summary of the invention
Purpose of the present invention just provides the technology of a kind of efficient and easy production rhKGF-2.
Another object of the present invention provides expression vector and the host cell that is used for this method.
In a first aspect of the present invention, a kind of dna sequence dna of coding human body keratinized cell growth factor-2 of optimization is provided, its dna sequence dna is shown in SEQ ID NO:1.
In a second aspect of the present invention, a kind of expression vector is provided, it contains the above-mentioned preferred hKGF-2 encoding sequence of the present invention (comprising dna sequence dna or RNA sequence).
In another preference, described expression vector is pET30a (+)/rhKGF-2 that has inserted sequence shown in the SEQ ID NO:1.
In a third aspect of the present invention, a kind of host cell is provided, it contains the dna sequence dna of the coding human body keratinized cell growth factor-2 of above-mentioned expression vector of the present invention or described optimization.More preferably, described host cell is intestinal bacteria.
In a fourth aspect of the present invention, a kind of method of producing human body keratinized cell growth factor-2 is provided, it comprises step:
(a) under the condition that is fit to the expressing human body keratinized cell growth factor-2, with the above-mentioned host cell of fermentor cultivation the present invention;
(b) from culture, isolate human body keratinized cell growth factor-2.
In another preference, described expression vector is expression vector pET30a (+)/rhKGF-2 that contains IPTG inductive promotor, and perhaps described host cell is intestinal bacteria.
In another preference, in step (a), carry out high density fermentation.
In another preference, described step (b) comprises step:
(i) broken fermentation thalline, the bacterium supernatant liquor is broken in centrifugal acquisition;
(ii) by saltouing and/or ultrafiltration is carried out preliminary purification to broken bacterium liquid supernatant liquor;
(iii) chromatography purification, described chromatography purification is selected from: affinity chromatography, cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography and combination thereof.
In another preference, described culture condition is: culture temperature is 30-40 ℃, more preferably 34-38 ℃; PH is between 6～8, more preferably between 6.5～7.5; Glucose or glycerol concentration are 0.1～5%, more preferably 0.3～3%; Inductor is 1mM IPTG, induces preceding OD 600Between 0.5～20, more preferably between 1～10; Induction time 0.5～10 hour, more preferably 1～5 hour.
Description of drawings
Fig. 1: the sequence alignment figure that has shown people KGF-2 native sequences and majorizing sequence of the present invention.The homogeny that shows both is lower, and about 70%.
Fig. 2: the structure wiring diagram of expression vector pET30a (+)/rhKGF-2.
Fig. 3: the enzyme of recombinant expression vector pET30a (+)/rhKGF-2 is cut evaluation.Each swimming lane is as follows: 1, and 5:DNAMarker (New England company); 2:pET30a (+)/KGF-2 plasmid; 3:pET30a (+)/KGF-2 cuts through the PstI enzyme; 4:pET30a (+)/KGF-2 cuts through the NdeI+SalI enzyme.
Fig. 4: the SDS-PAGE electrophorogram of different substratum.Each swimming lane is as follows: 1: the molecular weight standard product; 2: before inducing; 3～6:M9 substratum (induces 1,2,3,4hr); 7～9:M9-4 substratum (induces 3,2,1hr).
Fig. 5: high density fermentation electrophorogram.1: the molecular weight standard product; 2, before the inducing of 5:1 number and No. 2 jar; 3, the 4:1 jar induce 2,3hr; 6, the 7:2 jar induce 2,3hr.
Embodiment
The inventor by the series of optimum to the hKGF-2 encoding sequence, and through screening, has obtained a majorizing sequence (SEQ ID NO:1) that is particularly suitable for escherichia coli expression through extensive and deep research in a plurality of modification sequences.This majorizing sequence not only selected colibacillary preferred codon for use, also eliminated some and has been unfavorable for the secondary structure expressed, therefore help prokaryotic cell prokaryocyte efficiently, the rhKGF-2 that stably expresses.Through the intestinal bacteria that transform, expression amount can reach about 800mg/L.Finished the present invention on this basis.
In another preference, to express with preferred carrier pET30a (+), this carrier helps correct fold of rhKGF-2 eukaryotic gene in protokaryon, and exists with soluble form, thereby has simplified separation purifying technique.
In another preference, the intestinal bacteria (abbreviating " engineering bacteria " as) that the hKGF-2 encoding sequence of optimizing with the present invention transforms, cultivate through high density fermentation, IPTG induces, rhKGF-2 is present in matrix with solvable, activity form, and easy purifying process of while is by two easy step purifying methods, can obtain purity and reach 95% pure product, obtain high expression level, high yield, have the method for a kind of rhKGF-2 of production of industrialization value.
The cDNA sequence of hKGF-2 is known, and its maturation protein is made up of 170 amino acid, and theoretical molecular is 19.3kD.The aminoacid sequence of maturation protein is shown in SEQ ID NO:2.
The inventor is earlier according to the e. coli codon preferences, cDNA sequence to hKGF-2 under the prerequisite that does not change its aminoacid sequence is optimized, also eliminate some then and be unfavorable for the secondary structure (as being unfavorable for hairpin structure of transcribing etc.) expressed, through test, in numerous modification sequences, obtained a special encoding sequence of optimizing.The nucleotide sequence of the coding hKGF-2 of this optimization is shown in SEQ ID NO:1, and is lower with the homology of hKGF-2 native sequences, only about 70% (see figure 1).The hKGF-2 mature polypeptide that the hKGF-2 encoding sequence of optimization of the present invention is still encoded natural, i.e. 170 amino acid whose protein of total length (SEQ IDNO:2).
Inventive point of the present invention mainly is to change the brand-new gene design of the hKGF-2 of structure.The technology such as plasmid, transformed into escherichia coli cell, the screening of engineering bacteria, the fermentation of engineering bacteria and the separation of product of inserting as for the hKGF-2 gene that will change structure are carried out (people such as Sambrook for example by ordinary method known in the art basically, molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)) or according to the method described in the Chinese patent application CN02110808.0.
Carrier of the present invention comprises expression plasmid, for example contains the plasmid expression vector of IPTG inductive promotor, and has inserted the encoding sequence of human body keratinized cell growth factor-2 (rhKGF-2) in described expression vector.A kind of particularly preferred expression vector is carrier pET30a (+), pSE280, pSE380 and pSE420.
Be applicable to that host bacterium of the present invention is a prokaryotic host cell, especially intestinal bacteria (E.coli).
Usually, 5 ' end in the KGF-2 preferred sequence adds initiator codon and terminator codon respectively with 3 ' end, and introducing specificity restriction enzyme site, be cloned into then in the expression plasmid that contains IPTG inductive promotor, transform the e. coli host bacteria compatible, after Screening and Identification, obtain engineering bacteria with expression vector.Engineering bacteria of the present invention belongs to the IPTG induction type.
In the present invention, the fermentation condition of engineering bacterium expression rhKGF-2 is not particularly limited.Can adopt the fermentation condition of this area routine.Usually, engineering bacteria is with peptone, yeast powder, NH 4Cl or (NH 4) 2SO 4Deng being nitrogenous source, glycerine, glucose, lactose etc. are carbon source, and VITMAIN B1 in the basic medium of VITAMIN, is shaken in the bottle or in the fermentor tank and cultivated, expresses as a supplement.
In addition, add each nutritive ingredient by control, factors such as control cultivation, induction period dissolved oxygen (DO), pH, temperature, time, nectar degree, specific growth rate can further improve expression amount.
For the selection of substratum, generally include the selection of nitrogenous source, carbon source, inorganic salt, VITAMIN, trace element etc.
(a) nitrogenous source contains organic nitrogenous source and inorganic nitrogen-sourced, wherein organic nitrogen source such as peptone, yeast powder, or the mixture of the two, total concn 0.1-3%; Inorganic nitrogen-sourced as ammoniacal liquor or NH 4Cl, (NH 4) 2SO 4Deng ammonium salt, concentration 0-1.5%.
(b) inorganic salt comprise phosphoric acid salt, Chinese holly hydrochloride, Mg 2+Salt etc.Preferably, phosphate buffer concentration 20-200mM, pH5-8; Mg 2+Salt 0-5mM.
(c) in substratum, add VITMAIN B1 VITAMIN as a supplement, concentration 1～1000PPM.
(d) according to M 9Trace element formula in the substratum, trace element can add 0.1-2ml/L training liquid.
(e) carbon source comprises glycerine, glucose, lactose etc.Can be single carbon source, also can be mixed carbon source.Preferably, carbon source is selected from down group: glycerol concentration is 0.1-5%; Lactose concn is 0.1-3%; Glucose concn is 0.1-5%.
For inductor, can use IPTG to make inductor separately; For inducing temperature, inducing temperature 30-40 ℃, preferably 34-38 ℃, more preferably about 37 ℃.
For the control of dissolved oxygen (DO), cultivation stage is more than 30% usually, and induction period is more than 50%.
Behind fermentation expression rhKGF-2, the rhKGF-2 that expresses is carried out routine separate.
Usually, fermented sample is earlier removed fermented liquid supernatant in modes such as centrifugal, filtrations, obtains thalline.Behind the buffered soln suspension thalline, carry out broken fully bacterium, obtain broken bacterium liquid in modes such as ultrasonic disruption, high-pressure machinery fragmentations.Then broken bacterium liquid is carried out centrifugal to remove bacterial chip, the broken bacterium liquid supernatant liquor of acquisition.Broken bacterium liquid supernatant contains target protein matter rhKGF-2.Broken bacterium liquid supernatant can by saltout, method such as ultrafiltration carries out carrying out chromatography purification again behind the preliminary purification, also can directly carry out chromatography purification.
Be applicable to that chromatographic technique of the present invention comprises cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, affinity chromatography etc.
(a) affinity chromatography:
Select heparin affinity gel medium, salt gradient or heparin gradient elution.
(b) anion chromatography.
Through 2-3 step purifying, can obtain the pure product of rhKGF-2, the purifying yield is more than 40%, and purity is more than 95%.In the present invention, by the optimization of hKGF-2 encoding sequence, the hKGF-2 expression amount can be risen from 200mg/ and bring up to 800～1000mg/ liter.
RhKGF-2 can make various formulations with routine techniques behind the purifying.
In an example of the present invention, selected to help the plasmid vector of eukaryotic gene correct translation in prokaryotic cell prokaryocyte: the expression vector that contains IPTG inductive promotor, and in described expression vector, inserted the encoding sequence of rhKGF-2, made up rhKGF-2 engineering bacteria (intestinal bacteria) efficient, stably express.Cultivate through high density fermentation, IPTG induces, and rhKGF-2 is present in the born of the same parents and matrix with soluble form; The expression level height accounts for bacterial protein about 50%, and the rhKGF-2 expression amount reaches 800～1000mg/L fermented liquid.Because expression level is higher, rhKGF-2 has natural radioactivity again, has avoided complicated renaturation process, by easy, two step purifying methods fast, promptly obtain the pure product of high-quality rhKGF-2, every liter of fermented liquid can get the pure product 300mg of rhKGF-2, and product purity is more than 95%.
Stoste adds suitable auxiliary material behind the purifying, makes the powder ampoule agent for injection of rhKGF-2.The preliminarily stabilised test shows that this powder injection is stable.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid can obtain the about 300mg of the pure product of rhKGF-2 or more, therefore is fit to industrialization production.
The invention has the advantages that:
(1) expression process is simple.
(2) expression amount height.By controlling crucial technological condition for fermentation, make expression level reach 800mg/L.
(3) purifying process is easy, rate of recovery height.Because albumen is to express with soluble form, has therefore simplified purification procedures, and the purifying rate of recovery is improved greatly, makes scale operation rhKGF-2 become possibility.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
RhKGF-2 expresses the structure of engineering bacteria
1. goal gene is synthetic
Natural acid sequence according to known hKGF-2, according to the e. coli codon preferences and consider to eliminate hairpin structure etc. and be unfavorable for the secondary structure expressed, under the condition that does not change aminoacid sequence, the encoding sequence (shown in SEQ ID NO:1) of design hKGF-2.Entrust Shanghai to give birth to the synthetic designed hKGF-2 majorizing sequence of worker's biotechnology company limited.
During the complete sequence of synthetic recombinant human keratinized cell growth factor-2 (rhKGF-2) gene, introduce the NdeI site and introduce Sal I site at 3 ' end at 5 ' end of this gene.
The structure of expression plasmid-pET30a (+)/rhKGF-2 with transform the host bacterium
Expression plasmid is pET30a (+) expression vector (this expression vector contains IPTG inductive promotor) available from invitrogen company, and the host bacterium is conventional e. coli bl21 (DE3).
Referring to Fig. 2.Handle gene rhKGF-2 and expression vector pET30a (+) respectively with NdeI and SalI double digestion, 37 ℃ of enzymes are cut and were reclaimed corresponding fragment in 3 hours and spend the night 16 ℃ of connections with the T4 dna ligase.Connect the product conversion and enter intestinal bacteria, select positive colony and carry out the plasmid enzyme restriction evaluation on the LB flat board that contains kantlex (100 μ g/mL): extractive plasmid DNA was reacted 3 hours at 37 ℃ with restriction enzyme PstI and NdeI+SalI, obtained having inserted in pET30a (+) recombinant plasmid pET30a (+)/rhKGF-2 of rhKGF-2 gene.
Utilize Pst I to carry out enzyme and cut evaluation.Pst I restriction enzyme site is present in KGF-2 gene inside, and this site not on the carrier.If during Nde I+SalI double digestion, can cut out the fragment of the about 500bp of size, just show that sequence correctly inserts among the carrier pET30a (+).
Enzyme is cut qualification result as shown in Figure 3.The result shows: behind the employing Pst I single endonuclease digestion (swimming lane 3), plasmid is the linearizing state, and size is in the position near 6000bp; In addition through behind the Nde I+SalI double digestion (swimming lane 4), can see fragment about the about 500bp of size from electrophoresis, above situation illustrates that the KGF-2 gene correctly inserts among the carrier pET30a (+).
In addition, with ordinary method just carrying out, reverse sequential analysis, confirm that cloned sequence and implementation sequence are in full accord.
Shake flask test, after this project bacterium was cultivated, after IPTG induced 2 hours, the target protein expression amount accounted for total protein about 30%.
Embodiment 2
The selection of suitable expression vector
According to embodiment 1 similar method, SEQ ID NO:1 is inserted several different expression vectors respectively, identical restriction enzyme site (NdeI/SalI) as pTrc/HisA (available from invitrogen company), pBAD/HisA (available from invitrogen company), pSE380 (available from invitrogen company), obtain plasmid pTrc/HisA/rhKGF-2, pBAD/HisA/rhKGF-2, pSE380/rhKGF-2.
With plasmid pTrc/HisA/rhKGF-2, pBAD/Hi sA/rhKGF-2, pSE380/rhKGF-2 transforms corresponding host bacterium respectively, picks out resistant strain.Carry out shake flask test with the engineering bacteria that contains plasmid pET30a (+)/rhKGF-2, after IPTG (or pectinose) induces 2 hours, the target protein expression amount that contains the engineering bacterium expression of plasmid pET30a (+)/rhKGF-2 accounts for total protein about 30%, and contain plasmid pTrc/HisA/rhKGF-2, pBAD/HisA/rhKGF-2, the target protein expression amount of the engineering bacterium expression of pSE380/rhKGF-2 accounts for total protein 15%, 18% and 20% respectively.
This shows that pET30a (+)/rhKGF-2 expression vector is better than other expression vectors.
Embodiment 3
Select optimal medium
Choose the e. coli bl21 that changes pET30a (+)/rhKGF-2 over to (DE3) (being designated hereinafter simply as " the engineering bacteria ") mono-clonal of preparation among the embodiment 1, be inoculated in the LB primary seed solution, cultivate 17-20hr; , in the 1L of 250ml LB Erlenmeyer flask, cultivate about 2～3hr in 1: 30 ratio two-stage inoculation, treat OD 600Reach 0.5～1 and promptly go up a jar fermentation (M9 substratum, improvement M9-2 substratum or improvement M9-4 substratum), pH6.8～7.2,37 ℃ of temperature, DO＞50% are treated OD 600Reach 3～4 IPTG that add 1mM and begin to induce, treat that pH (carbon source exhausts) back of rising adds 10% carbon source (glucose) and nitrogenous source feed supplement with suitable commentaries on classics degree stream and keeps pH 6.8～7.2, induces 3hr to finish.Sample carries out SDS-PAGE and detects (and scanning), measures protein content.
The determining the protein quantity result as shown in Figure 4.The result shows that M9 and M9-4 substratum all can be used for expressing hKGF-2, and the M9-4 substratum is better than M9.
In addition, also can obtain higher protein content with M9-2 culture medium culturing base.
Culture medium prescription
Name of material Required material Concentration Weighing
The LB substratum Peptone 1％ 0.5g
Yeast extract 0.5％ 0.25g
NaCl
1％ 0.5g
The M9 substratum NaCl 0.5％ 0.5g
MgSO 4 0.1％ 0.1g
Glucose 0.2％ 0.2g
NH 4Cl 0.1％ 0.1g
KH 2PO 4 0.3％ 0.3g
Na 2HPO 4.12H 2O 0.6％ 0.6g
Improvement M9-2 substratum Glucose 2％ 2g
Yeast extract 0.75％ 0.75g
K 2HPO 4 0.5％ 0.5g
KH 2PO 4 0.35％ 0.35g
(NH4) 2HPO4 0.55％ 0.55g
MgSO 4 0.2％ 0.2g
Trace element 0.1％ 0.1ml
Trisodium citrate 0.17％ 0.17g
Improvement M9-4 substratum CA 0.5％ 0.5g
K 2HPO 4·3H 2O 0.524％ 0.524g
KH 2PO 4 0.2％ 0.2g
(NH 4) 2SO 4 0.12％ 0.12g
Anhydrous MgSO 4 0.05％ 0.05g
NH 4Cl 0.02％ 0.02g
Glycerine 0.1％ 0.1g
Na 2HPO 4·12H 2O 0.7％ 0.7g
CaCl 2 1ml/L 0.1ml
Embodiment 4
The research of engineering bacteria high density fermentation
Choose the engineering bacteria mono-clonal, be inoculated in the high-density improvement M9 substratum seed liquor incubated overnight, OD 600Reach 1～4 and promptly go up a jar fermentation (high-density improvement M9 substratum), control pH7.0,37 ℃ of temperature, DO＞50% begin flow feeding behind about 3～4hr, treat OD 600Reach the IPTG that adds 1mM about 10 and begin to induce, every 1hr keeps sample, and induces 3hr to finish.Sample detection SDS-PAGE (and scanning), mensuration protein content.
Protein content the results are shown in Figure 5.The result shows, utilization high density fermentation technology is optimized the output of KGF-2, it is extremely successful that the result proves, on the basis that expression level does not descend substantially (about 30%), owing to improved the nectar degree greatly, make heavy further the reaching more than the 25g of wet bacterium of every liter of training liquid, the target protein expression amount is brought up to 800～1000mg/L training liquid, has improved the production efficiency of hKGF-2 greatly.
Embodiment 5
The purifying of rhKGF-2
4 ℃ of centrifugal 10min of following 5000rpm of fermented liquid get thalline, and the thalline height crushes bacterium, and the centrifugal 30min of 8000rpm gets supernatant.Ultrafiltration and concentration and exchange buffering liquid: use the Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 10KD, leaves and takes concentrated solution (effect is to remove small molecular weight impurity and salt) during ultrafiltration.Fermented liquid supernatant ultrafiltration to volume is left about 500ml, and (PH7.4 20mM), continues ultrafiltration to add PBS; This program repeatedly, (PH7.4,20mM) conductivity water of damping fluid flushes closely until the gentle PBS of the conductivity water of sample.
1. chromatography 1 (affinity chromatography):
Chromatography media: heparin
Damping fluid: solution A: PBS (PH7.4,20mM)
Solution B: PBS (PH7.4,20mM)+1M NaCl
Last sample: with sample on the rhKGF-2 solution of ultrafiltration and concentration.
Clean: clean chromatography column with the solution A of 6CV after going up sample.
Gradient: clean the back and solution B is risen to 100% from 0% with 10CV.
Collect: collect rhKGF-2 sample peak.
2. chromatography 2 (anion chromatography)
Chromatography media: Q Sepharose FF
Damping fluid: solution A: PBS (PH7.4,20mM)
Solution B: PBS (PH7.4,20mM)+1M NaCl
Last sample: with sample on the rhKGF-2 solution.
Clean: clean chromatography column with the solution A of 6CV after going up sample.
Gradient: clean the back and solution B is risen to 100% from 0% with 10CV.
Collect: collect rhKGF-2 sample peak.
Sample is through this two steps purifying, and promptly after affinity chromatography, the anion chromatography, purity is increased to more than 95%.
With the MTT determination of color hKGF-2 of routine promotion proliferative effect to the NIH3T3 cell (l cell) of routine.The result shows that the hKGF-2 behind the purifying has the promotion proliferative effect that promotes the NIH3T3 cell, and active identical with commercially available hKGF-2.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
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Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys
100 105 110
Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser
115 120 125
Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly
130 135 140
Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser
145 150 155 160
Ala His Phe Leu Pro Met Val Val His Ser
165 170

Claims (10)

1. the dna sequence dna of the human body keratinized cell growth factor-2 of encoding is characterized in that, its dna sequence dna is shown in SEQ ID NO:1.
2. an expression vector is characterized in that, it contains the described dna sequence dna of claim 1.
3. expression vector as claimed in claim 2 is characterized in that, described expression vector is pET30a (+)/rhKGF-2 that has inserted sequence shown in the SEQ ID NO:1.
4. a host cell is characterized in that, it contains the dna sequence dna of described expression vector of claim 2 or the described coding human body keratinized cell growth factor-2 of claim 1.
5. host cell as claimed in claim 4 is characterized in that described host cell is intestinal bacteria.
6. method of producing human body keratinized cell growth factor-2 is characterized in that it comprises step:
(a) under the condition that is fit to the expressing human body keratinized cell growth factor-2, with the described host cell of fermentor cultivation claim 4;
(b) from culture, isolate human body keratinized cell growth factor-2.
7. method as claimed in claim 6 is characterized in that, described expression vector is expression vector pET30a (+)/rhKGF-2 that contains IPTG inductive promotor, and perhaps described host cell is intestinal bacteria.
8. method as claimed in claim 6 is characterized in that, carries out high density fermentation in step (a).
9. method as claimed in claim 6 is characterized in that, described step (b) comprises step:
(i) broken fermentation thalline, the bacterium supernatant liquor is broken in centrifugal acquisition;
(ii) by saltouing and/or ultrafiltration is carried out preliminary purification to broken bacterium liquid supernatant liquor;
(iii) chromatography purification, described chromatography purification is selected from: affinity chromatography, cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography and combination thereof.
10. method as claimed in claim 6 is characterized in that, described culture condition is: culture temperature is 30-40 ℃, more preferably 34-38 ℃; PH is between 6～8, more preferably between 6.5～7.5; Glucose or glycerol concentration are 0.1～5%, more preferably 0.3～3%; Inductor is 1mM IPTG, induces preceding OD 600Between 0.5～20, more preferably between 1～10; Induction time 0.5～10 hour, more preferably 1～5 hour.
CNB2005100272064A 2005-06-28 2005-06-28 Production process of recombinant human horny cell growth factor-2 Expired - Fee Related CN100487118C (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925647A (en) * 2016-06-24 2016-09-07 成都远睿生物技术有限公司 Fermentation method for recombinant human keratinocyte growth factor-2 engineering bacteria and fermentation medium

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925647A (en) * 2016-06-24 2016-09-07 成都远睿生物技术有限公司 Fermentation method for recombinant human keratinocyte growth factor-2 engineering bacteria and fermentation medium

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