CN1521183A - Method for high flux refolding renaturation of denaturation recombinant protein - Google Patents

Abstract

The present invention relates to high-flux refolding renaturing method for denatured recombinant protein. The renaturation of insoluble denatured protein inclusion body as material is renatured in an artificial triple molecular mate-detergent-anticoagulant system. The renaturation process includes making the inclusion body become extending structure state with the system to form protein-detergent composite; diluting with urea or guanidine buffering liquid to 10-50 times and adding urea or guanidine buffering liquid containing molecular mate and oxidation and reduction system to release extending protein structure gradually; and purifying with parallel separating column refolded and renatured recombinant protein before concentration or freeze drying. The present invention has the advantages of high density of protein to be renatured, small reaction container, validity to inclusion body or denatured protein formed in various expression system, etc.

Description

A kind of denaturation recombination protein high-throughput refolding renaturation method

Technical field

The present invention relates to the refolding renaturation of denatured protein (inclusion body), specifically a kind of denaturation recombination protein high-throughput refolding renaturation method.

Background technology

The one's last year in 20th century, the finishing of the Human Genome Project, the industrialization of life science with swift and violent speed great-leap-forward entered brand-new era, i.e. genome times afterwards comprehensively; The various new branch of science of genome project, cross discipline is arisen at the historic moment, and development of biology is just as the water in the Changjiang river, and is at a tremendous pace, form a macroscopic and be referred to as " life science industry ", and not only be confined to biotechnology or technical field of bioengineering; Yet the essence of life is protein, and when recognizing the mankind only less than 40,000 genes, many scientists make unremitting effort and creation for exploring this essence.Although, the existing nearly 4000 kinds of recombinant proteins in the whole world are produced, but the protein that can be used for directly expression and preparation is less than 25%, the recombinant protein that most gene engineering bacteria (intestinal bacteria or fungi) is expressed is that inclusion body (Inclusion Body) form as sex change secretes cell, exists with undissolved condensation product.This just makes and obtains the bottleneck that a large amount of recombinant human functional proteins become the life science industrial development.Therefore, the high throughput protein separating and purifying technology of exploitation milligram level and even gram level becomes a big focus of current life science industry member.

The escherichia coli prokaryotic expression system has become the daily work of biology laboratory or biotech company, and its reason is: intestinal bacteria cheaply are easy to get, and protein synthesis is efficient, and the technology cost is more relatively cheap and efficient than the newborn animal of dried meat, insect and fungal systems; Some are had cytotoxic protein, can obtain a large amount of recombinant proteins fast by this system, overexpression does not damage for intestinal bacteria; Reorganization back insoluble protein is stable, is easy to preserve; Can avoid the degraded of the expressed recombinant protein of other system, problems such as instability.Utilize classical cell wall breaking (N,O-Diacetylmuramidase or mechanical process, lysozymeor French Press), centrifugal (10, the 000xg low-speed centrifugal), again through freeze thawing and the inclusion body (Krueger that can obtain sex change with different buffering washing lotion washing methods efficiently, J.K. wait Biopharm, 1989,3:40-45).Can more easily obtain to be higher than the insoluble protein inclusion body of 90% purity like this; Because numerous gene molecule library commercializations, it is standby to set up recombinant human protein inclusion body molecular library fast with this conventional method like this.Its key is the refolding renaturation technology of denatured protein (inclusion body), accomplishes that especially high-throughout refolding is that industry member is badly in need of one of difficult problem that solves.

Protein folding is a complicated process, and the interior environment of keeping proteinic three-dimensional structure and cell in the body has much relations, can be the adjusting of molecular chaperones or other auxiliary substances; Yet, external refolding method, except using conventional thermodynamics regulation mechanism (as controlling concn, pressure, pH value etc.), common refolding renaturation method comprises: dilution method and osmose process.But, in many cases,, usually be difficult to take active protein because the intermediate product of folding renaturation more easily forms aggregate (Aggregates); Recently by turning on the folding simulation of body internal protein, produced some new methods, for example: use molecular chaperone protein matter GroEL has the effect of conjugated protein in vivo, is equivalent to the affinity ligand of denatured protein; Immobilization GroEL post (fixed bed) is equivalent to the affine adsorption chromatography post of denatured protein, thereby can improve the treatment capacity of sample, and make protein in renaturation, obtain concentrating and purifying, its characteristics are: the recycling problem that 1) not only can solve molecular chaperones, and because the utilization (being bordering on plug flow) of fixed bed, can improve the utilising efficiency of molecular chaperones; 2) owing to adopt operate continuously, the raw material treatment capacity is big, and product can be concentrated, purifying; 3) be easy to set up relevant model, have important directive function for amplifying to produce; Teshima etc. utilize the immobilized molecules companion, at external folding renaturation (the .Appl Microbiol Biotech such as Teshima T that has assisted amylase, carbonic anhydrase, DNA enzyme, 1997,48:41-46), the molecular chaperones that is applicable to protein refolding that other receive much concern also comprises: protein two sulphur are good for isomerase (polypeptide proline(Pro) cis-trans isomerase, Peptidyl Proly Cis-Trans Isomerase, be called for short PPI), GroEL, GroES, DnaJ, DnaK; Also have report to utilize " mini-chaperone " that target protein is carried out renaturation (.Proc Nail Acad Sci USA such as Zahn R, 1996,93:15024-15029), " mini-chaperone " is meant the fragment of GroEL, and this fragment is not the hydrolysate of GroEL, but by the expression product of gene fragment in intestinal bacteria that comprises GroEL191-345 amino-acid residue fragment (16.7kD) or 191-376 amino-acid residue fragment (21kD) password after the gene recombination, they can promote cyclophilin A effectively, the renaturation of rhodanese and barnase; Because " mini-chaperone " molecule is less, be more suitable in immobilization, and do not need to add in addition renaturation cofactor (as GroES and ATP etc.), therefore have great application prospect; But,,, still belong to expensive reagent for extensive multiple proteins preparation because these molecular chaperoneses all need special technology of preparing.

Summary of the invention

The object of the present invention is to provide low, the regulatable denaturation recombination protein high-throughput refolding renaturation method that is suitable for the mass-producing application of a kind of cost.

For achieving the above object, the technical solution used in the present invention is:

With inexpensive artificial molecule companion system " three-in-one " method, with insoluble denatured protein (inclusion body) (or the commercial enzyme of using) is raw material, adopt artificial molecule companion-stain remover-dispersion stabilizer three renaturation systems to separate purification of recombinant proteins matter, specific operation process is as follows:

1) under 4～36 ℃ of temperature, (method is referring to Babbit, Biotechnology such as P.C. 1990,8:945-949 preferably will repeatedly to wash inclusion body (purity＞90%) behind the purifying; Wong, H.H. wait Bioseparation 1996,6:185-192) make inclusion body become the stretching, extension attitude of primary structure with 6-8M urea or 3-7M guanidine damping fluid, wherein contain protein wt 0.1～0.5% (w/v) stain remover and 0.4～0.6M dispersion stabilizer, 0.5 after～5 hours, form stretching, extension attitude protein-stain remover mixture and add stain remover and dispersion stabilizer in denatured protein solution, by forming complex body with protein, the inhibiting peptide interchain is assembled mutually; (ultra-violet analysis detects A to add stain remover ₂₈₀) to there not being the free attitude protein that stretches;

2) add 10～50 times of 1～2M urea or guanidine damping fluid dilutions, making protein concn is 0.1～10 mg/ml, 0.5～2 hour time (being diluted to desirable renaturation concentration with renaturation solution); Decide according to concrete protein, 80% inclusion body is for going back ortho states, for the known inclusion body that contains non-reduced attitude sulfydryl, can add excessive gsh in buffered soln as weak reductant, be in phthalin with the inclusion body of keeping all unfoldings; Remove when the purifying inclusion body, it is the same with cell walls important fully to remove membranin, and keeping and going back ortho states is the key that obtains the renaturation recombinant protein matter of high yield;

3) adding contains 1～2M urea or the guanidine damping fluid that concentration is 20～100mM (millimolar concentration) molecular chaperones and redox system, progressively discharges the protein structure that stretches attitude, and renaturation in 3～5 hours time needed for～1 week individually; Impulse method control (can in 90～120 minutes intermittent type or gradient type) adds the damping fluid speed that contains molecular chaperones, progressively discharge the prlmary structure of protein that stretches attitude, keep suppressing simultaneously false folding and inclusion body regeneration, the protein that making dissociates stretches attitude concentration and reaches ideal, is beneficial to the concentration of intramolecular reaction; Control primary structure protein becomes the partially folded inactive state PFP that does not promptly contain the stain remover molecule from the speed that stain remover-mixture strips down, and further finishes the renaturation of correct unfolded protein, thereby is folded into natural active protein NP.

4) use the recombinant protein of various parallel separator column purifying refolding renaturations, concentrate or lyophilize (with protein soln or solid mode, respectively at 4 ℃ ,-20 ℃, or-80 ℃ of storages);

Described molecular chaperones is: alpha-cylodextrin, beta-cyclodextrin or methyl-beta-cyclodextrin.

Inclusion body unfolding urea damping fluid consists of: 6-8M urea, 1-5mM EDTA (disodium salt disodium), 0.1M 2-Me, 20mM NaOAc; The guanidine damping fluid consists of: the sodium phosphate of 3-7M guanidine, 150mM, 25mM DTT (dithiothreitol (DTT)), 10mM MgCl ₂With 10mM KCl, pH5-7.6.The stain remover and the dispersion stabilizer that can add 0.1-0.5% in the damping fluid simultaneously: 0.4-0.6M arginine, 0.05% polyoxyethylene glycol (3550-5000 molecular weight)

Described stain remover is a sodium laurylsulfonate, hexadecyl front three ammonia hydrogen bromide doped quaternary ammonium salt (CTAB), trimethyl-glycine or straight chain cyclodextrin; Described dispersion stabilizer is L-arginine, polyoxyethylene glycol (3500～6000), glycerine, glucose, glycine, calcium lactate, zinc chloride, proline(Pro) or thetine (NDSBs); Described redox system is meant and contains 10～100mM Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), reductive agent contract sulphur ethylene glycol, mercaptoethanol, 1～30mM halfcystine, 1～30mM glutathione or dithiothreitol (DTT), 10: 1～1: 1 gsh/half gsh, 10: 1～1: 1 Guang ammonia/half light ammonia or 10: 1～1: 1 Gelucystine/halfcystine in the urea damping fluid; (actual conditions folds/condenses than deciding according to each protein properties) its consumption is that 5～100 times of protein equivalent concentration are excessive; Described parallel separation and purification post is that stationary phase is that Saphodex 30 is to Sophadex 300 gel-filtration columns or reversed phase chromatography post; Flow velocity=5～8.4 milliliter/hour; Described parallel separation and purification post products therefrom can be further purified by the fast protein liquid chromatography method; Described after adding molecular chaperones, employing is at 4 ℃ of constant temperature renaturation methods or adopt the great-jump-forward temperature-raising method to improve the refolding renaturation yield, promptly earlier 4 ℃ of renaturation to maximum activity, be warming up to 20～36 degree then, can shorten renaturation time several times, reduce the formation that protein active lost or condensed by product in the long-term renaturation process, improve the purity of recombinant protein matter; Gained purification liquid can reuse ultrafiltration and concentration to respective concentration, or adds stablizer and make concentrated solution in-20 ℃ of storage (activity can keep the several months), or lyophilize one-tenth contains the dry powder that is lower than 1～2% moisture content, in-80 ℃ of prolonged preservation (more than 1 year).

Described inclusion body raw material can be obtained by following method:

1) inclusion body of introducing human body exogenous gene expression/synthetic recombinant protein in intestinal bacteria, be settled out the inclusion body of corresponding gene fast respectively, set up corresponding particle inclusion body crude product library (method: Krueger .Biopharm such as J.K. 1989,3:40-45);

2) the purification inclusion body to higher purity (Marston, Methods of Enzymology such as F.A.O, 1990,182,264-276), then, use 6～8M urea buffer solution to make inclusion body become the stretching, extension attitude of primary structure.

Wherein: " three-in-one " method or title " cocktail " method (stain remover+cyclodextrin+dispersion stabilizer or short folding agent) is a main models, (Bao Dao " two-in-one " method was referring to auxiliary carbonic anhydrase and egg albumen lysozyme renaturation in the past, Rozema D et al.Biochemistry, 1996,35:15760-15771), and our " temperature jump technology " (4 ℃ 30-36 ℃ then of delivering in early days, Xie, Y. etc., D.B.Protein Science 1996,5 (3): 517-523).

The present invention has following advantage:

1. the present invention uses the combinatorics principle to carry out the parallel renaturation of protein, recombinant protein matter concentration height, and used reaction vessel volume is little, and it is all effective to carry out parallel refolding renaturation for the formed inclusion body of various expression systems; By regulating ratio, feed way (in proper order) and the feed rate of artificial molecule companion cyclodextrin, stain remover and dispersion stabilizer, reach the technology of the parallel protein purification of thermokinetics coordinated regulation, can obtain multiple recombinant protein fast; Improve tens of times than rapid dilution method and dialysis method efficient.α or methyl-beta-cyclodextrin with cheapness are the model molecular chaperones, set up a regulatable refolding platform technology of " cocktail " method (CART) that can be suitable for efficient protein matter renaturation; It is the industrialization platform method that required function protein in the proteomics research is provided a large amount of preparations.

The present invention by with escherichia expression system as model, set up the technology of high-throughput denatured protein (inclusion body) refolding renaturation of general other any expression systems.Compare with protein molecule companions such as GroE, stain remover-cyclodextrin-dispersion stabilizer unite use, promptly so-called " cocktail " collaborative refolding technology (CART) of regulating has tangible advantage: (1) artificial molecule companion does not belong to protein, be difficult for affected by environment and inactivation, operational condition is comparatively gentle; (2) stain remover and cyclodextrin all can directly be bought, and save the step of macromole molecular chaperones separation and purification; (3) molecular weight of stain remover and cyclodextrin is less, and easy and protein separation helps improving industrial production efficiency, and refolding and ultrafiltration and concentration can " be treated different things alike "; Can make and separate purification inclusion body efficient and improve several times and even tens of times; This method all has effect preferably to proteolytic enzyme (Protease), matrix metalloproteinase (MatrixMetalloprotease) and kinases preparations such as (Kinase), and method is easy, is applicable to the protein group engineering research.

3. refolding reaction of the present invention can be carried out in a reactor, the trouble of having avoided intermediate material to shift, and the optimization of proteinic refolding process simultaneously can be judged as follows and not measure active:

1) process does not have a large amount of precipitations to form;

2) protein can be by molecular weight mwco membrane in the protein renaturation device, ultrafiltration and concentration to 20～70 mg/ml (A280 nanometer spectrophotometry), and micromolecular compound and renaturation agent can be removed by this method;

3) one of column chromatography generation is folding unimodal;

4) the judicious molecular weight of irreducibility SDS-gel electrophoresis;

5) protein can be more definite by protein pattern in crystallization or the use protein library.

High-throughput prepares the active recombinant protein that has more than the milligram level, and method is easy, and condition optimizing is easy, and industrialization prospect is good; Concrete optimal conditions depends on:

1) method of renaturation system: pulse liquid feeding or multistep processes; One step liquid feeding or gradient liquid feeding method, stable state liquid feeding method and jump temperature-raising method;

2) purified proteinic inherent physico-chemical property;

3) collocation of the component in the damping fluid of refolding renaturation and further gel column separation and purification, the protein after the refolding is purified by gel filtration column chromatography or high-efficient liquid phase technique.

Description of drawings

Fig. 1 is the schematic diagram of denatured protein of the present invention " cocktail " regulation and control refolding renaturation technology (CocktailAligned Refolding Technology-CART).Wherein: non-unfolded protein UP:(Unfolded Protein); Middle transition attitude UP-D:(Unfolded Protein-DetergentComplex); Partially folded thing PFP:(Partial Folded Protein); Condensation product Ag (Aggregates) natural protein NP (Native Protein).

Fig. 2 is an operational flowchart of the present invention.

Embodiment

Embodiment 1

The refolding renaturation of HIV-1 proteolytic enzyme D30W: promptly HIV-trypsinase inclusion body is (50 milligrams, U.S. BiotaiX, Inc.) be dissolved into (100mM Tris in the 8M urea, pH3.5), the concentration dilution of urea is to 2M (pH3.5,1mM DTT and 3% PEG, 0.2% C-12 SDS then, 0.5M arginine monohydrochloride), the protein ultimate density is 0.5 mg/ml, stirs 1 hour, adds alpha-cylodextrin damping fluid (Aldrich then, 6 equivalents, 1～2mM reduced glutathion: GSH, the gsh of 0.1-0.2mM oxidized form: GSSG), stirred 5 hours, ultrafiltration (molecular weight is held back ultra-filtration membrane=10,000) in the molecule thickener of design voluntarily.After being concentrated to 5 milliliters, regulate PH to 4.4 (adding about 0.5 milliliter of sodium-acetate buffer that contains the 100mM of 1M sodium-chlor); After 20 minutes, at room temperature centrifugal 20 minutes (10 of protein soln, 000g), reject a small amount of insolubles, supernatant liquor (PH3.5,4 hours) is behind gel filtration chromatography, concentrate once more through homemade bio-reactor again, get respective egg white matter solution (yield 35%), purity＞90% (SDS-PAGE and HPLC analyze), stand-by 4 ℃ of storages.

For obtaining maximum efficiency and renaturation yield, can utilize homemade parallel reactor (national patent application number: 03210921.0).

Embodiment 2

HIV-1 proteolytic enzyme V82N refolding renaturation: promptly HIV-proteolytic enzyme is (50 milligrams, U.S. BiotaiX.) molten in the 8M urea, the concentration dilution of urea is to 2M (pH3.5 then, 1mM DTT and 3% polyoxyethylene glycol), protein ultimate density 0.5 mg/ml, stirred 1 hour, add stain remover and dispersion stabilizer damping fluid (0.2%SDS, 0.5M arginine monohydrochloride, 100mM sodium-acetate, pH4.5,1mM DTT and 1% PEG), stirred 1 hour, add alpha-cylodextrin (Aldrich then, 5 equivalents, 1-2mM reduced glutathion (GSH), the gsh of 0.1-0.2mM oxidized form (GSSG) stirred 5 hours, the sucking-off reaction solution, centrifugal 20 minutes (10,000g), supernatant liquor (about 100 milliliters) voluntarily the design the molecule thickener in ultrafiltration (molecular weight is held back ultra-filtration membrane=10,000) be concentrated to 5 milliliters after, regulate PH to 4.4 (adding about 0.5 milliliter of sodium-acetate buffer that contains the 100mM of 1M sodium-chlor); After 20 minutes, protein soln is recentrifuge 20 minutes (10 at room temperature, 000g), reject a small amount of insolubles, supernatant liquor concentrates in homemade molecule thickener once more through gel filtration chromatography, gets respective egg white matter solution, concentrate back mensuration and contain 38 milligrams of active proteins (purity＞90%) approximately, stand-by 4 ℃ of storages.

Embodiment 3

Preparation (the preparation method Ido of inclusion body of HIV-1 variant protein matter D30F, E. wait J.Biol.Chem.1991,266,24359-24366.): it is molten that (the 100mM sodium-acetate PH4.5), stirred 2 hours in the 8M urea, use 2M urea damping fluid (PH3.5 then, 1mM DTT and 3% PEG, 0.2%SDS, 0.5M arginine monohydrochloride), be diluted to final protein concentration 0.5 mg/ml, stirred 1 hour, and added alpha-cylodextrin damping fluid (Aldrich, 5 equivalents thereafter, contain 1-2mM reduced glutathion (GSH), 0.1 the gsh of～0.2mM oxidized form (GSSG)) stirred 5 hours sucking-off reaction solution, centrifugal 20 minutes (10,000g), the sodium-acetate and the 1mM DDT that add 400 milliliters of 10mM in the supernatant liquor, PH5.0, the time kept 5 hours.10,000g is centrifugal, removes insoluble substance.This Trypsin matter solution is concentrated to 5 milliliters with the molecular weight mwco membrane.Proteinic concentrated solution is measured concentration in A280 nanometer (E0.1%=1.0).Use A Pu to force the protein active of HIV-1 inhibitor titration determination institute of medicine company above 90%.

Embodiment 4

The renaturing inclusion bodies of recombinant C aditropsinogen: the Caditropsinogen inclusion body is according to currently known methods (Lin .J.Biol.Chem.1992 such as X., 267,17257-17263) preparation.50 milligrams of inclusion bodys are dissolved in 2 milliliters and contain 8M urea damping fluid (50mM Tris, 1mM 0.8 and 1mM EDTA, 100mM 2-Me, pH8.0,5.7mM CATB, 0.5M arginine), stirred 30 minutes, centrifugal (175,000Xg) 30 minutes, remove insolubles, use stain remover damping fluid (containing ultimate density 2M urea) to be diluted to protein concn 1.0mg/ml then, stirred 1 hour, in final solution, add methyl-beta-cyclodextrin damping fluid and (contain 6 normal methyl-beta-cyclodextrins, 1～2mM reduced glutathion: GSH, 0.1 the gsh of～0.2mM oxidized form: GSSG, 50mM Tris-HCl), impulse method intermittent type three times reinforced (about 1 hour), stirred 48 hours at 4 ℃ then, with 10,000Da molecular weight mwco membrane, ultrafiltration and concentration to 10 milliliter, centrifugal (175,000xg) removed not tolerantly on a small quantity in 15 minutes, (3 * 90cm post is used 20mM Tris-HCl to supernatant liquor with Sephacryl S-300 column chromatography, damping fluid balance and the wash-out of pH7.0,25 milliliters/hour of flow velocitys), active part is with FPLC (anion-exchange column momoQ is 7.0 20mM Tris-HCl damping fluid balance with pH), use 0～1MNaCl linear gradient elution again, active constituent solution is after desalination, and lyophilize gets 7.5 gram protein, 15% yield, SDS-PAGE analyzes, and shows unimodal.Purity is greater than 90%.

Embodiment 5

The refolding renaturation of reorganization propepsin (Pepsinogen): (preparation method is referring to Lin for the inclusion body particle of purifying, X. wait J.Biol Chem 1989,4482-4489) (50 milligrams) are through centrifugal (16,000xg, 30min) be dissolved in the 6M urea and (contain the 100mM 2 mercapto ethanol, 60 milliliters of volumes), 4 ℃ were stirred 6 hours.Residual insoluble substance is removed through centrifugal (286,000xg, 2 hours), be diluted to 200 milliliters of (100mM Tris-HCl, 0.3% thetine (NSBAs), 2mM EDTA with the urea damping fluid, 0.4M arginine monohydrochloride, 5mM GSH, 0.5mM GSSG, 0.1mM PMSF, pH8.0), stirred 2 hours, and added alpha-cylodextrin (100mM α-CD, 6 equivalents, 1～2mM reduced glutathion: GSH, 0.1 the gsh of～0.2mM oxidized form: GSSG, 50mM Tris pH8.0) stirred 5 hours, this solution is through molecular weight mwco membrane 30,000 ultrafiltration and concentration to 10 milliliter, centrifugal (286,000xg) 1 hour, supernatant liquor Sephacryl S-300 gel filtration chromatography, 3 * 90-cm post 20mM Tris-HCl, pH7.5, flow velocity 30/ hour.If use fast protein liquid chromatogram (FPLC) purifying (Pharmacia) anionresin MonoQ HR 5/5 post.Damping fluid is 20mMTris-HCl, pH8.0 solution gradient 0-1M NaCl.30 minutes, 1 ml/min.After the desalination, dry, corresponding recombinant protein matter (15mg, renaturation yield 30%, purity＞95%).

Embodiment 6

Recombinant protein streptokinase (StreptoKinase, SK) purifying and expression, this recombinant protein express from pET11 genophore BL21 (DE3) the intestinal bacteria bacterium colony that (method is referring to Studier, Methods Enzymol.1990 such as F.W., 185,60-89.).SK (SK residue 1-147).Expressed inclusion body is pressed example 1 described condition refolding after purifying.50 milligrams of inclusion bodys, after the renaturation, further at Sephacryl S-300 post (Pharmacia Biotech, Uppsala Sweden) goes up purifying, and elution buffer is 20mM Hepes and 0.4M urea, pH7.5, itself after anion-exchange resin column (20mM Hepes and 0.4M urea, pH7.5,0～0.1M NaCl are eluent.Yield 15%, purity＞90%.

Embodiment 7

Recombinant human neurenergen 3 (Human Neurotrophin-3, renaturation NT-3).The recombinant human neurenergen 3 is expressed (Suenaga by report side, M. wait Biotechnology Appl.Biochem1998,28,119-124), 10 gram frozen cells (wet basis) are dispersed in (10mMEDTA in 40 milliliters of damping fluids, pH7.0) ultrasonic cell wall breaking, (17,000g) one hour, supernatant liquor discarded suspended centrifugal, the inclusion body particle is dispersed in the above-mentioned damping fluid again, and is centrifugal.The journey twice that repeats to correct one's mistakes guarantees to remove all cells rubbish, and membrane proteolytic enzyme.The inclusion body particle is with 30 milliliters of 4M urea damping fluids (50mMTris-HCl, pH8.0,5mM DTT) homogenate then, and recentrifuge (17,000g) 60 minutes.The inclusion body particle of final purifying 8M urea damping fluid (60 milliliters, pH3.0,20mM Trisodium Citrate, 0.1%SDS) behind the middle unfolding, stirred 2 hours, 10 times (three subpulses are reinforced, the 50mM beta-cyclodextrin to add the renaturation solution dilution, 0.1M sodium phosphate, 0.2 arginine, 1.0mM GSH and 0.2mMGSSG, pH8.5), after 4 ℃ of one weeks of stirring, adopting jumps is rapidly heated to 12-20 degree method, stirs 3 hours, and activity reaches maximum (＞90%, former method needs one month), renaturation solution is concentrated to 10 milliliters, with SP-Sepharose (Pharmacia Biotech, Sweden, 10 * 150cm, contain 0.1%CHAPS, the 0.1M sodium phosphate buffer of pH6.0 makes column equilibration) column chromatography, elutriant is the same damping fluid that contains 0.4M NaCl (pH6.0), gained stream part is through RP/HPLC (ODS-120T, TosohCorporation, Japan) post, with the 20-36% acetonitrile wash-out that contains 0.1% TFA, gained stream part gets 10 milligrams of NT-3 through lyophilize.Purity＞95%.

Embodiment 8

Carbonic anhydrase II (CAC AB II, Sigma, Inc., 20 milligrams) be dissolved in 5 milliliters of 6M salt and calculate the guanidine damping fluids and (contain 50mM Tris, 1mM DTT, pH8.5), 4 ℃ with 2M urea damping fluid (pH7.5,1mM DTT and 1% PEG, 1.1mM CTAB, 0.5M arginine monohydrochloride, 1.43mMEDTA, 5.7mM CTAB, 5.7mM GSH, 0.57mM GSSG, pH7.5,50mM Tris) dilution is 10 times, stirred 2 hours, and added methyl-beta-cyclodextrin (6 equivalent methyl-β-CD, 1～2mM reduced glutathion: GSH then, 0.1 the gsh of～0.2mM oxidized form (GSSG), 50mMTris, twice intermittent type adds), stirred 3 hours, ultrafiltration and concentration (molecular weight mwco membrane 20K Da), light scattering method detects, and renaturation yield 97% uses Guanidinium hydrochloride damping fluid repeatability method stable yield (Xie in earlier stage than us, Y. wait .Protein Science 1996,5 (3): 517-523).

Embodiment 9

Matrix metalloproteinase catalytic site (the protMMP3 that blocks, polypeptide 1-255, MW=28,779) preparation: express apo-MMP3 inclusion body (Marcy according to currently known methods, A.I. wait Biochemistry 1991,30,6476-6483.), 50 milligrams of protMMP3 inclusion bodys are dissolved in (50mM HEPES in the 8M urea, 0.1M NaCl), stirred 1 hour, with 2M urea damping fluid (50mM HEPES, pH7.5,0.1M NaCl, 10mM CaCl2,0.2 SDS, 0.5M arginine, 30mM EDTA, 4mM 1, the 10-phenanthrolene) be diluted to 0.4 mg/ml protein concn, add isopyknic 50mM HEPES, pH7.5, and 0.1M NaCl.), stirred 1 hour, add methyl-beta-cyclodextrin (5 equivalents at 4 ℃, 1～2mM reduced glutathion: GSH, 0.1 the gsh of～0.2mM oxidized form: GSSG, 50mM HEPES, pH7.5, add in three batches), with damping fluid (50mM HEPES, pH7.5,0.1M NaCl, 2mM 1,10-phenanthrolene, and 3mM EDTA) stirs after 5 times of the dilutions and spend the night ultrafiltration then, with (5 milliliters of damping fluids, 50mM HEPES, pH7.5,0.1M NaCl) dilution back sucking-off centrifugal (12,000g), take out supernatant liquor, discard insolubles, cryodesiccated 10 milligrams are removed prothetic group protMMP3 catalytic site (10 milligrams).The gained kinase inactive, activation recovering behind the adding zine ion.This method can avoid oneself protein hydrolysis phenomenon (related activity analysis and Wetmore, D.R.Biochemistry 1996,35, method therefors such as 6549-6558 with).

Embodiment 10

The purification process of reorganization arginine kinase: arginine kinase (AK) inclusion body obtains (Strong ﹠amp according to standard method; Ellington, Comp Biochem Physiol 1996,113B, 809-816), Bacillus coli cells is through the N,O-Diacetylmuramidase broken wall, and after the freeze thawing, (10,000g) centrifugal, inclusion body is with 80mL damping fluid IV (50mM Tris pH8.0 on Beckman JA12 whizzer; 2% bent X-100 (TritonX-100); 10mM EDTA; The 1M urea) washs with the weeding of grease plastid protein of nucleic acid and some other pollution.Same method, the washing three times after centrifugal AK be distributed to damping fluid (50 milliliters, 10mM TrisHClpH8.0; 1mM EDTA; 1mM DTT; 10mM NaCl, and 0.02%w/v NaN ₃), under the room temperature, the sex change unfolding is 1 hour in the 8M urea, it is centrifugal then that (8,000xg) 1 hour BeckmanJA20 whizzer is by 0.22 μ m membrane filtration, again through gel filtration chromatography (2.5 * 120 centimetres of Sephacryl S-300 posts), and elutriant 6M urea (50 milliliters, 10mM TrisHCl pH8.0; 1mMEDTA; 1mM DTT; 10mM NaCl, and 0.02%w/v NaN ₃), behind the ultrafiltration and concentration, get and be equivalent to 50 milligrams of AK inclusion body concentrated solutions, with sex change liquid 8M urea (50mM TrisHCl, 10mMEDTA, 2mM DTT) be diluted to 0.5 mg/ml, under 4 ℃ of conditions, slowly add 2M urea renaturation solution (100 milliliters contain 50mM TrisHCl, pH8.0 through peristaltic pump; 1mM EDTA, the 100mM2-mercaptoethanol, 0.1%SDS, 0.5M arginine monohydrochloride, 1mM DTT and 1% PEG) in, stir and formed AK-stain remover mixture in 2 hours, add alpha-cylodextrin (1～2mM reduced glutathion (GSH), the gsh of 0.1-0.2mM oxidized form (GSSG), 50mM Tris-HCl then, pH8.0, three times intermittent type adds 6 equivalents, 3 equivalents/time) remove stain remover, stirred renaturation 5 hours, centrifugal 20 minutes (10,000g), through anion exchange chromatography (2.5 * 120cm Sephacryl S-100 post, dress post naturally, UV detects, 10 to 60mM KCl gradient elution (10mM Tris, pH8.0,1mM EDTA, 1mM DTT, and 0.02%NaN ₃) the product of purifying, vacuum ultrafiltration in molecule thickener (10kDa) is concentrated to～1mL at last, both the arginine kinase (10 milligrams) of purifying.SDS-PAGE shows purity＞90%.

Embodiment 11

The renaturation of ox pancreas RNase: ox pancreas RNase A is 50 milligrams of RNase of commercial enzyme (Sigma) in the damping fluid of 6M Guanidinium hydrochloride (2 milliliters, 0.1M Tris-HCl, pH8.0,2mM EDTA, 0.14DTT, 0.4M arginine, 0.2%CTAB) stir 18 hours unfolding inactivation (Lyles under the room temperature, M.M. wait Biochemistry 1991,30,613-619).(use 0.1M Tris-HCl, pH8.7 contains 1M Guanidinium hydrochloride and 1mM EDTA to this solution, and (protein concentration is measured at 278nm, and the inactivation coefficient is 9300M to 1M to remove DTT and to reduce the concentration of Guanidinium hydrochloride by Sephadex G-25 short column ^-1Regulate the reductive enzyme, regulate anaenzyme concentration to 1mg/mL, stirred 2 hours with 1M Guanidinium hydrochloride (containing 0.1M Tis-HCl, pH8.7,0.5M arginine, 0.2%CTAB, 1mM EDTA).Add methyl-beta-cyclodextrin (6 equivalents, 1～2mM reduced glutathion: GSH, 0.1 the gsh of～0.2mM oxidized form (GSSG), 0.1M Tris-HCl, three subpulse formulas add), (enzymic activity is measured by following method: cCMP was dissolved in 0.05mM Tris-HCl damping fluid (pH7.5) and contains 5mM EDTA, cCMP concentration (0.12 mg/ml) in 5 hours 30 ℃ of stirrings.Get in the supernatant liquor (100 μ L) that cCMP solution (2.4 milliliters) is dissolved in renaturation, in the absorption of 30 ℃ of wavelength 287 nanometers test cCMP solution, with 100%RNase A specific activity), renaturation yield＞90%.

Embodiment 12

Human Lymphotactin (Human Lymphotactin, hltn) be to become that unique one group of specificity attracts T-lymphocyte and natural killer cell protein in the medicine cytokine C class: this protein is made up of 93 residues, does not have 2 or the halfcystine of 4-position reservation.The inclusion body of hltn can make by currently known methods (Kuloglu, Biochemistry such as E.S 2001,40,12486-12496), promptly 1 liter of cell that fermented liquid obtained is stuck with paste and is scattered in 50mMTris-HCl, pH8.0,50mM NaCl, 50mM PMSF, 2mMEDTA), through after the freeze thawing repeatedly, add CaCl ₂The Snase (Aldrich-Sigma) of activation in the fused protein to ultimate density be 10mM.Stirred 20 minutes, centrifugal (20000xg) 15 minutes rejects supernatant liquor, lower floor's particle broken wall damping fluid (0.5%Triton-100) washed twice of 50mL.Get corresponding inclusion body after centrifugal, get 50 milligrams and be dissolved in 7M Guanidinium hydrochloride damping fluid (40mMTris-HCl, pH7.4,20mM EDTA, 50mM DTT, 0.2%CTAB, 0.5M arginine), stirring at room 3 hours, centrifugal (20000g) 20 minutes, supernatant liquor takes out, (contain 100mM Tris-Ac with renaturation solution 1M Guanidinium hydrochloride, pH8.0,0.2%CTAB, 0.5M arginine, 25 μ M 2-MTT) be diluted to 0.5 mg/ml, 4 ℃ were stirred 3 hours, added alpha-cylodextrin (5 equivalents, the adding of three subpulse formulas, 1～2mM reduced glutathion (GSH), 0.1 the gsh of～0.2mM oxidized form (GSSG), 100mM Tris-HCl, pH8.0), stirred 16 hours, ultrafiltration and concentration (3000Da molecular weight mwco membrane) with getting ionized water (50 milliliters) washing three times, is concentrated to about 1 milliliter then then to 10 milliliters then, with decomposing fused protein damping fluid (0.5M Tris-HCl, pH8.0,1M NaCl, 10mM CaCl ₂) dilute 10 times (every milliliter contains 5 milligrams of warm protein approximately).This fused protein solution was handled 1～2 day with Xa factor (2 units/10 milligram warm protein, Xa factor is available from Promega), this hltn product, through reversed-phase HPLC (the C18 post, Vydac, Hesperia, CA USA) separates and makes.Lyophilize gets 5 milligrams of protein, and purity is greater than 95% (SDS-PAGE shows that its molecular weight is 10.3kDa, and is same with reported method).

This method is not limited to the listed proteolytic enzyme of this patent, matrix metalloproteinase and kinases, also is not limited to colibacillus engineering.Suitable equally to the inclusion body that other expression systems obtained.

Claims (8)

1. denaturation recombination protein high-throughput refolding renaturation method, it is characterized in that: the inclusion body with insoluble denatured protein is a raw material, adopt artificial molecule companion-stain remover-dispersion stabilizer three renaturation systems to separate purification of recombinant proteins matter, specific operation process is as follows:

1) under 4～36 ℃ of temperature, inclusion body with the washing purifying, use 6～8M urea or 3～7M guanidine damping fluid, make inclusion body become the stretching, extension attitude of primary structure, wherein damping fluid contains 0.1～0.5% stain remover and 0.4～0.6M dispersion stabilizer, 0.5 after～5 hours, form and stretch attitude protein-stain remover mixture;

2) add 10～50 times of 1～2M urea or guanidine damping fluid dilutions, making protein concn is 0.1～10 mg/ml, 0.5～2 hour time;

3) adding contains 1～2M urea or the guanidine damping fluid that concentration is 20～100mM artificial molecule companion, 4～6 equivalents and redox system, progressively discharges the protein structure that stretches attitude, and the time was 3 hours～1 week; Described molecular chaperones is: alpha-cylodextrin, beta-cyclodextrin or methyl-beta-cyclodextrin

4) recombinant protein of the parallel separator column purifying refolding renaturation of use concentrates or lyophilize;

2. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: inclusion body unfolding damping fluid consists of: contain 1～5mM EDTA in 6～8M urea; PH4～8,0.1M 2-Me; The sodium phosphate that contains 150mM in 3～7M Guanidinium hydrochloride, PH 7.6; 25mM DTT, 10mM MgCl ₂With 10mM KCl.

3. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: described stain remover is sodium laurylsulfonate, trimethyl-glycine, polytrimethylene ether or straight chain cyclodextrin.

4. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: described dispersion stabilizer is L-arginine, polyoxyethylene glycol, glycerine, glucose, glycine, calcium lactate, zinc chloride, proline(Pro) or thetine.

5. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: described redox system is meant and contains 10～100mM Tri(Hydroxymethyl) Amino Methane Hydrochloride, reductive agent contract sulphur ethylene glycol, mercaptoethanol, 1～30mM halfcystine, 1～30mM glutathione, dithiothreitol (DTT), 10: 1～1: 1 gsh/half gsh, 10: 1～1: 1 Guang ammonia/half Guang ammonia or 10: 1～1: 1 Gelucystine/halfcystine in the urea damping fluid; Its consumption is that 5～100 times of protein equivalent concentration are excessive.

6. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: described parallel separation and purification post is that stationary phase is that Saphodex 30 is to Sophadex 300 gel-filtration columns or reversed phase chromatography post; Flow velocity=5～8.4 milliliter/hour.

7. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: described parallel separation and purification post products therefrom can adopt the fast protein liquid chromatography method to be further purified.

8. according to the described denaturation recombination protein high-throughput of claim 1 refolding renaturation method, it is characterized in that: described after adding molecular chaperones, employing is at 4 ℃ of renaturation constant temperature renaturation methods or adopt the great-jump-forward temperature-raising method to improve the refolding renaturation yield, both elder generation to maximum activity, was warming up to 20～36 degree 4 ℃ of renaturation then.

Images