整理GST融合蛋白纯化方法

1、精品文档GST 融合蛋白纯化方法 Purification of GST Fused ProteinsAbstract:Many people have ven ted out frustratio n over in soluble GST-fused prote ins. This is a protocolfor en zymatically active soluble GST-fused protei ns. All GST-fused prote ins are ren dered soluble with this tech nique though en zyme activi

2、tiy can range from 30-90%.Materials and Reage nts1. STE Buffer10 mM Tris-HCl, pH 8.01 mM EDTA150 mM NaCl2. Lysozyme soluti on10 mg/ml in water (make fresh)3. PBS4. Elution Buffer50 mM Tris.Cl, pH 9.020 mM GSH5. 10% Sarkosyl in STE Buffer6. 10% Triton X-100 in STE Buffer7. 1 M DTT8. 100 mM IPTGProced

3、ureDay 11. Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin.Day 21. Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicilli n.2. Grow at 37 C to an A600 of 0.6 to 0.8.3. Induce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr at 37C or grow overnight at room tempera

4、ture.Lower IPTG concen trati ons and lower grow ing temperatures tend to produce greater solubility at the expe nse of yield.4. Pellet cells by cen trifugi ng at 3000g, 4 oC for 10 min. Deca nt media and resuspe nd cells in 30 ml ice-coldPBS to wash. Tran sfer to a 40-ml Oak Ridge tube and cen trifu

5、ge at 3000 g, 4oC for 10 min. Deca nt PBS.5. This is a convenient point to stop and to store pellets at -80oC. Else continue to lyse cells.6. Thaw pellet on ice if cells are froze n else proceed to the n ext step.7. Resuspend pellet in 10 ml of ice cold STE Buffer.8. Add 100 ml of freshly prepared l

6、yozyme solution, incubate on ice for 15 min. Just before sonication, add 100 ml of 1 M DTT and 1.4 ml of 10% Sarkosyl. Mix thoroughly by in version and son icate for a total time of 1 mi n.9. Cen trifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Tran sfer super nata nt to a50-ml co n

7、ical tube and discard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20ml. The effective concen trati on of Sarkosyl and Trit on X-100 will be 0.7% and 2% respective ly. In cubate at room temperature for 30 min.10. Pour the lysate to 1 ml bed of prepared Glutathi one Sepharos

8、e in PBS. In cubate at room temperature for 30 min to 1 hr with agitatio n.To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, in vert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and

9、 resuspe nd beads with 1 ml of PBS.11. Wash the beads with 3 X 50 ml of PBS. Fi nally resuspe nd in 5 ml of PBS. Pour to adispo-colu mn. Wash the 50-ml con ical tube with an additi onal 5 ml of PBS. Pool with the first 5 ml in the dispo-colu mn.To wash, use the same centrifugation technique for prep

10、aring the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick to pipette tips.12. If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions with SDSPAGE精品文档精品文档亲和层析实验技术方法INTRODUCTIONThis protocol describes a method for removi ng

11、an tibodies that react with bacterially en coded protei ns by passing a crude preparation of immunoglobulins through a column containing immobilizedbacterialprotei ns.MATERIALS* Reage ntsE. coli stra in used as host for preparati on of expressi on libraryAn tibody preparati on that is to be used for

12、 scree ningThis protocol works best when using an IgG fraction, prepared by chromatography of the antiserum on prote in A-Sepharose. Cell lysis buffer0.1 M sodium borate (pH 8.0)1 M NaClSterilize the cell lysis buffer using a 0.45-m filter, an cjk store at room temperature. Approximately 100 mlof ce

13、ll lysis buffer is required per 1 liter of bacterial culture.* Growth mediumOne liter of growth medium appropriate for the E. coli strain of choice is required.* LysozymeDissolve solid lysozyme at a concen trati on of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately beforeuse. Make sure that the pH of

14、 the Tris solution is 8.0 before dissolving the protein. Lysozyme will not work efficiently if the pH of the solution is less than 8.0.Use a molecular biology grade of lysozyme. Add solid lysozyme to assist lysis of bacterial cells.* NaOH (1 N)The preparation of 10 N NaOH involves a highly exothermi

15、c reaction, which can cause breakage of glass contain ers. Prepare this soluti on with extreme care in plastic beakers. To 800 ml of H2O, slowlyadd 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. Whenthe pellets have dissolved completely, adjust the volu

16、me to 1 liter with H2O. Store the solution in a plasticcontainer at room temperature. Sterilization is not necessary.* Pan creatic DNase Io 1 mg/ml Pan creatic DNase Io 50 mM NaClo 10 mM Tris-Cl (pH 7.5)o 1 mM MgCl 2Dissolve 2 mg of crude pan creatic DNase I (Sigma or equivale nt) in 1 ml of 50 mM N

17、aCl, Tris-Cl (pH 7.5),1 mM MgCl 2. When the DNase I is dissolved, add 1 ml of glycerol to the solution and mix by gently 精品文档inverting the closed tube several times. Take care to avoid creating bubbles and foam. Store the solutionin aliquots of -20C dd solid DNase I to the bacterial cell lysate to d

18、igest chromosomal DNA.Tris-buffered Sali ne (TBS) Dissolve 8 g of NaCl, 0.2 g of KCI, and 3 g of Tris base in 800 ml of distilled H2O. Add 0.015 g ofphe nol red and adjust the pH to 7.4 with HCl. Add distilled H 2O to 1 liter. Dispe nse the solution into2aliquots and sterilize them by autoclavi ng f

19、or 20 minu tes at 15 psi (1.05 kg/cm) on liquid cycle. Store thebuffer at room temperature.* TBS containing 0.2% (w/v) sodium azide4 Triton X-100METHODGrow a 1-liter culture of the appropriate strain ofE. coli (e.g., Y1090hsdR, XL1-Blue, or DH1) tostati onary phase.* Recover the bacteria by cen trif

20、ugati on at 4000g (5000 rpm in a Sorvall GSA rotor) for 20 minu tes at 4C. Pour off the medium, and sta nd the cen trifuge tubes in an in verted positi on to allow the last traces of medium to drain away.* Resuspe nd the pellet in 100 ml of Cell lysis buffer.* Add 200 mg of lysozyme, and in cubate t

21、he bacterial suspe nsion for 20 minu tes at room temperature.* Add 1 mg of pa ncreatic DNase I and 200 l of Triton X-100.* Incubate the bacterial suspensionfor 1 hour at 4 C,or until the turbidity clears and the viscositydecreases.* Cen trifuge the bacterial lysate at 8000g (8200 rpm in a Sorvall SS

22、-34 rotor) for 20 minu tes at 4C.Carefully deca nt the super nata nt into a fresh flask.+ Adjust the pH of the supernata nt to 9.0 with 1 M NaOH.* Determine the concentration of protein in the lysate using the Lowry, Bradford, or other method of measureme nt.* Chill the extract to 0C, and then bind

23、the bacterial proteins to cyanogen-bromide-activated Sepharose 4B or to Affi-Gel 10 accord ing to the manu facturers in struct ions.* Before use, equilibrate the Sepharose 4B or Affi-Gel 10 resi n containing conjugated E. coli protei nsin TBS containing 0.2% (w/v) sodium azide.* Use 1 ml of settled

24、volume of resi n coupled to E. coli an tige n for each milligram of IgG protein to be purified by affinity chromatography. Mix the IgG and the coupled resin and incubate for 12-18 hours at room temperature on a rotati ng wheel device.Load the slurry into a chromatography column. Recover the antibody

25、 by washing the column with TBS.Collect fracti ons (0.2 colu mn volume each) un til the OD280 drops to zero. Pool the fractions containing an tibody, and store the pool at -20 C un til it is used for immuno logical scree ning.REFERENCES精品文档精品文档1. de Wet, J.R., Fukushima, H., Dewji, N.N., Wilcox, E.,

26、 OBrien, J.S., and Helinski, D.R. 1984. Chromogenic immunodetection of human serum albumin and alpha-L-fucosidase clones in a human hepatoma cDNA expression library. DNA 3: 437-447.MedlineAnyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no r

27、epresentations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these materia

28、l, please consult:Molecular Cloning: A Laboratory Manual, Third Edition, Joseph Sambrook and David W. Russell, ? 2001by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p.14.28-14.30.精品文档GST融合蛋白纯化方法1目的片段接入pGEX载体；2涂板，挑单克隆，摇菌至OD60B 1.0，加入IPTG （终浓度1 mM）诱导6 8 h ;3收菌，每升菌液约以 50 mL PBS重悬，

29、加入1%Triton X-100 （v/v ）, 1% 3 -巯基乙醇（v/v ）, PMSF（终浓度 1 mM）;以下步骤均在冰上操作：4超声破碎菌体，15000 g , 10min离心取上清，在上清中加入适量GST-beads,轻轻晃动令其吸附蛋白1 h;5 2000 g , 3min离心弃上清；6加入至少10倍体积PBS轻摇至beads悬浮于溶液中，2000 g , 3 min离心弃上清；7重复步骤6两次；8 力口入 1 mL GST Elution Buffer，轻摇 10 min ；9 2000 g , 3 min离心，收集上清；10重复步骤8-9至少两次；11 SDS-PAGE电泳

30、检测蛋白纯度，Bradford法检测蛋白浓度；12将蛋白置于-20C保存。P.S.大量提取前应取少量菌液，改变IPTG浓度，诱导温度，诱导时间等，以确定蛋白表达的最适条件。GST fusi on protein purification GST融合蛋白纯化1) grow 20ml cells O/N37C dilute 50X into prewarmed LB,grow to 0.6 OD or about 1hr.Induce w/2mM IPTG (238mg/0.5l),grow 3hr,spin 6k GS3 10,freeze at 70C2) extract cells (fro

31、m 500ml)i n 25 mls.Hei ntz Buffer plus trito n (HBT)by gen tle pipette resuspe nding on ice circa 10after thaw ing.3) tra nsfer to 50ml coni cal ss34 flip top tubes.4) add 10mg lysozyme powder to the 50ml (cells from 1 liter now in 1 50ml tube),digest 15 on ice.5) sonicate with large probe 1 80% pow

32、er,freeze in IN,thaw at37C sonicateaga in on ice,solutio n should become viscous.6) add 1mg DNAse and RNAse,incubate on ice 15.7) spin 7.5k rpm4 C ss34 10.8) tra nsfer super nata nts to coni cal screw caps,freeze in IN2,may store.9) Batch adsorb w/ 4ml 50% slurry GT-Sepharose (PL 17-0756-01),1hr,4C

33、spin 2,4k on bench.10) aspirate,resuspe nd in 25ml HBT,sp in, repeat.11) pour slurry into column (Econo 1.7x20),elute to top,then with 20 column vols.of HB-T.12) elute protein in minimal volume (5-10ml)HB 5mM GT (Sigma G4251,1.5 mg/ml).13) lN 2 freeze as 100ml aliquots for GS.HB 1 literFi nal Stock

34、ml/l25mM HEPES,pH7.9 1M 501mM EDTA,pH 8.0 0.5M 220% glycerol stock 2001mM MgCl 21M 160mM KCl 2M 301% Trit on stock 10 add before use0.5mM DTT 1M 0.5 0.5mM PMSF 0.5mM 10 5g/ml Leupeptin 5mg/ml dilute before use5mg/ml an tipa in ” ”check pH!GUSHISTOCHEMICAL STAINING1) determ ine nu mber of slides n ee

35、ded,multiply by 0.75ml2) make up required vol of sta in:for 10ml4 C 5mg X-Gus50 d nn dimethyl formamide,dissolve10ml 50mM NaPO 4 pH 73) sect ions best cut with vibrat ing knifefor sect ions w/ chlorophyll,put i n cell- wells w/ 500 d l sta infor sect ions w/out chlorophyll,put directly on slides w/

36、sta in4) inc o/n37 C in humidity chamber5) asp,i nc 10in FAA:for 200ml 4 C 10ml formaldehyde10ml HAc75ml EtOHH2O vol6) i nc 2 50% EtOH7) i nc 2 100% EtOH8) inc 1 H 2O精品文档融合蛋白纯化，方法Purification of GST fusion proteins in E.coli GSTMak ing GST fusi on protei ns:(07/19/03)ver.1Grow up 5ml LB with Amp o/n

37、.Add to 45ml LB with Amp37o shake 2.5 - 3 hrs,till OD 600 0.4-0.8Put bottles in room temperature water for 10 min to cool dow n.Add 100 卩 l 0.2M IPTG to 0.4mM finalShake 30 C 2hrPellet bacteria,deca nt sup,i nvert to drainResuspe nd in 1ml NETN 0.2mM PMSF / 50ml LBPMSF,stock 10mMNETN: 20mM Tris-Cl (

38、pH8)100mM NaCl1mM EDTA0.5% NP40store at 4 CVortex to mix wellSoni cate at scale 5 for 15sec.Keep on ice.For 10ml Corning tubes,use scale 7Spin 4 C ,5minTran sfer super nata nt to a new tube.To each lysate,add 60 卩 l 50% GlutathioneSepharose 4BPepette 400 卩 l Sepharose stock (75%)Spin 1000rpm 5min, d

39、iscard super nantantWash 3x300 卩 l NETNResuspend in 300 卩 l NETN to get 50% beadsMix in cold room for 2 hours,slowly whirlPellet beads by brief cen trifugati on, carefully discard super nata ntWash 3x1ml NETN/PMSFWash 2x1ml Elution Buffer (50mM Hepes,pH7.9,40mM KCl,1mM EDTA 1mM DTT)Elute proteins by

40、 mix beads with 60卩 l eachEluti on buffer5mM Glutathio ne,(for 10mM,use 3.07mg/ml)1mM DTTSlowly swirl at RT 1hrQuick spin to pellet,tra nsfer super nata nt to a new tubeRe- elute with 60 卩 l eachNETN精品文档精品文档5mM Glutathio ne1mM DTTSlowly swirl at RT 30mi nQuick spin, comb ine super nata nt,sp in and

41、tran sfer super nata nt twice to avoid any residual beads.total is 120 卩 wn oDialyze vs 50% glycerol/10mM Hepes,pH7.5/ 40mM KCI/ 1mM EDTA/ 1mM DTT/ 1mM PMSF in cold room for 2hr or o/n, store at -20CProte ins can also be concen trated in a Cen triprep-30 concen trator.The pore size of the membra ne

42、in the Cen triprep-30 allows glutathi one to pass into the aqueous compartme nt.PBS can be added to the prote in concen trate and the concen trati on procedure can be repeated.Thinking aliquot and save at -80 CRun 12% SDS-PAGEPurification of GST fusi on protei ns in E.coli GST融合蛋白纯化，方法二GST Protein P

43、rep.Ver.21) Grow 50ml of culture in LB or TB antibiotic o/n at 37 C shaker.2) Dilute culture in LB or TB an tibiotic 1:103) Grow 3hrs at 37 C .p l of 14) 1 nduce culture by addi ng 0.4 mM IPTG fin al co ncen tratio n.(For 50 ml fin al culture add 20 IPTG).5) Grow at 25 C for 1 hr.6) Spin 5 min at 5

44、K7) Wash pellet with half volume of cold H2O.(For 50 ml culture use 25 ml H2O.)8) Wash bacteria aga in.9) Resuspe nd in 1 ml of resuspe nsion buffer per 50 ml of culture.Resuspe nsion Buffer- NETN protease in hibitors20 mM Tris pH 8.0100 mM NaCl1 mM EDTA0.5% NP-40 or Trito n-X1g/ml Aproteinin1 p g/m

45、l PMSF1 p g/ml BenzaminideNote: Tim has 100x stock of protease in hibitors.10) S oni cate for 2x for 10 sec onds in cold room.精品文档11) Pellet debris by spinning at 4C (Easiest way is to put samples in multiple eppendofs and spinin cold room at max for 2 mi n.Binding of Fusion Protein to Beads1) Pipet

46、te 400卩 l of GSSepharose into Eppendof2) Spi n beads 15 sec 8,000 RPM3) Wash beads 2x with 4 C NETN4) Resuspend pellet in 320卩 l of NETN (Final volume will be 550卩 l)and put into 4 eppendofs.5) Add 300l of resuspension buffer and 75卩 l of E.Coli.GST Lysate to each tube.6) 1 ncubate for 30 min while

47、rock ing in cold room.7) Spi n 15 seco nds at 8,000 rpm.8) Wash 3x with 600卩 l of reuspension buffer9) Remove super nata nt and resuspe nd in appropriate volume resuspe nsion buffer.(You can addthis directly to SDS load buffer for running on a gel.)Purification of GST fusi on protei ns in E.coli GST

48、融合蛋白纯化，方法三，纯化小量Small scale fusi on prote in preparati onGrow 5ml culture o/n in TB with amp.Add o/n culture to 50ml of TB amp and grow for 3 hours in 37C shaker.Induce culture by adding 20卩 l of 1M IPTG (final 0.4mM)and transfer to 25 C shaker for 1hour.Pellet Bacteria 10min at 3KResuspe nd bacteria

49、 in 1ml of NETN protease in hibitors.Soni cate 2x for 5-10sec onds each time.Spin out cell debris by spinning in cold microfuge 5 min.at max.Remove supernatant and store at -70 C .Binding fusi on prote in lysate to beadsRemove 400 卩 l of GST beads into eppendorf and spin 15 sec at 8k.Remove super na

50、ta nt and wash 2x with NETN.Resuspe nd beads in 320 卩 l of NETN (550 卩 l fin al volume)Aliquot 50 卩 l of beads and add up to 200卩 l of GST lysate.If 200卩 l bring fin al vol.up to 200卩NETN.Incubate at 4 C with rocking for 30min.Wash 3x with NETN and add lysate containing protein of in terest.Incubate

51、 1-2hr at 4 C with rocking.Wash 3x with appropriate salt buffer.Boil proteins off in 2xSB for western blotting.精品文档NETN 2xSB20mM Tris pH8.0 125mM Tris pH6.8100mM NaCI20% Glycerol1mM EDTA 4.1% SDS0.5% NP-40 2%BME0.005% Bromphe nol BlueGST Purificati on Large Scale ProtocolResuspe nd 500ml pellet in 1

52、0ml of NETN and mix well (Keep at 4C)Combine two pellets (20ml)and transfer 20ml of resuspension to 50ml conical Soni cate 2x with 15 sec pulsesSpin for 20 at 10k in Sorvall at 4 C .Tran sfer super nata nt into new 50ml tubes.Add 1ml of washed beads rock for 30min at RTPour sample into colu mn and d

53、ra inSave flow through and repeat previous step 2xWash beads with 10ml of 1xPBS 3xAdd 500l of Elution buffer and rock for 10min at RTDrain and collect 500 卩 l of fractionRepeat 2x moreEluti on Buffer (5ml)NETN20mM Tris pH 8.010mM glutathio ne 15.6mg 100mM NaCl50mM Tris pH 8.0 125 M 1mM EDTA出0 4.9ml

54、0.5% NP-40精品文档融合蛋白纯化（方法四）筛选表达Purification of GST fusion proteins in E.coli GSTScree n of GST-Fusio n Protein Expressi on(pGEX system by Amersham: for check clones for expressi on of the desired fusi on protein prior to large-scale purificatio n)Pick several colonies of E.coli tran sformed with the p

55、GEX recomb inants into separate tubes containing 2 ml of 2 x YTA medium.-Note: For comparison,it is advisable to inoculate a control tube with bacteria transformed with the pare ntal pGEX plasmid.Grow liquid cultures to an A600 of 0.6-0.8 (35 h)with vigorous agitation at 2037C .Incubate fusion prote

56、in expression by adding 2卩 l of 100 mM IPTG (final concentration 0.1 mM).Continue incubation for an additional 12 hTran sfer 1.5 ml of the liquid cultures to labeled 1.5 ml microce ntrifuge tubes.Cen trifuge in a microce ntrifuge for 5 sec and discard the super nata nt.Resuspend each pellet in 300卩

57、l of -celd 1 x PBS,remove 10 卩 l of these resuspend cells intolabeled tubes (for later use in SDS-PAGE an alysis).-Note: Except where no ted,keep all samples and tubes on ice.Lyse the cells using the soni cator equipped with an appropriate probe.-Note: Lysis is complete whe n the cloudy cell suspe nsion becomes tran sluce nt.The freque ncy andintensity of sonication should be adjusted such that complete lysis occurs in 10 sec,without frothing (i