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1、GST融合蛋白纯化方法PurificationofGSTFusedProteinsAbstract: Many people have vented out frustration over insoluble GST-fused proteins. This is a protocol for enzymatically active soluble GST-fused proteins. All GST-fused proteins are rendered soluble with this technique though enzyme activitiy can range from
2、 30-90%. Materials and Reagents 1. STE Buffer 10 mM Tris-HCl, pH 8.01 mM EDTA150 mM NaCl 2. Lysozyme solution 10 mg/ml in water (make fresh) 3. PBS 4. Elution Buffer 50 mM Tris.Cl, pH 9.020 mM GSH 5. 10% Sarkosyl in STE Buffer 6. 10% Triton X-100 in STE Buffer 7. 1 M DTT 8. 100 mM IPTG ProcedureDay
3、1 1. Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin. Day 2 1. Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicillin. 2. Grow at 37oC 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 37oC or grow overnight at room temperatu
4、re.Lower IPTG concentrations and lower growing temperatures tend to produce greater solubility at the expense of yield. 4. Pellet cells by centrifuging at 3000 g, 4oC for 10 min. Decant media and resuspend cells in 30 ml ice-cold PBS to wash. Transfer to a 40-ml Oak Ridge tube and centrifuge at 3000
5、 g, 4oC for 10 min. Decant 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 frozen else proceed to the next step. 7. Resuspend pellet in 10 ml of ice cold STE Buffer. 8. Add 100 ml of freshly prepared lyozyme so
6、lution, 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 inversion and sonicate for a total time of 1 min. 9. Centrifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Transfer supernatant to a 50-ml conical tube and d
7、iscard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20 ml. The effective concentration of Sarkosyl and Triton X-100 will be 0.7% and 2% respectively. Incubate at room temperature for 30 min. 10. Pour the lysate to 1 ml bed of prepared Glutathione Sepharose in PBS. Incubate
8、at room temperature for 30 min to 1 hr with agitation.To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, invert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and resuspend beads with
9、1 ml of PBS. 11. Wash the beads with 3 X 50 ml of PBS. Finally resuspend in 5 ml of PBS. Pour to a dispo-column. Wash the 50-ml conical tube with an additional 5 ml of PBS. Pool with the first 5 ml in the dispo-column. To wash, use the same centrifugation technique for preparing the beads. When tran
10、sferring 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 SDS PAGE亲和层析实验技术方法INTRODUCTIONThis protocol describes a method for removing antibodies that react with bacter
11、ially encoded proteins by passing a crude preparation of immunoglobulins through a column containing immobilized bacterial proteins.MATERIALS ReagentsE. coli strain used as host for preparation of expression libraryAntibody preparation that is to be used for screeningThis protocol works best when us
12、ing an IgG fraction, prepared by chromatography of the antiserum on protein 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, and store at room temperature. Approximately 100 ml of cell lysis buffer is required per 1 liter of bac
13、terial culture. Growth mediumOne liter of growth medium appropriate for the E. coli strain of choice is required. LysozymeDissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately before use. Make sure that the pH of the Tris solution is 8.0 before dissolving the p
14、rotein. 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 exothermic reaction, which can cause breakage of glass con
15、tainers. Prepare this solution with extreme care in plastic beakers. To 800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the volume to 1 liter with H2O. Store the solution in a p
16、lastic container at room temperature. Sterilization is not necessary. Pancreatic DNase I o 1 mg/ml Pancreatic DNase Io 50 mM NaClo 10 mM Tris-Cl (pH 7.5)o 1 mM MgCl2Dissolve 2 mg of crude pancreatic DNase I (Sigma or equivalent) in 1 ml of 50 mM NaCl, Tris-Cl (pH 7.5), 1 mM MgCl2. When the DNase I i
17、s dissolved, add 1 ml of glycerol to the solution and mix by gently inverting the closed tube several times. Take care to avoid creating bubbles and foam. Store the solution in aliquots of -20C. Add solid DNase I to the bacterial cell lysate to digest chromosomal DNA. Tris-buffered Saline (TBS) Diss
18、olve 8 g of NaCl, 0.2 g of KCl, and 3 g of Tris base in 800 ml of distilled H2O. Add 0.015 g of phenol red and adjust the pH to 7.4 with HCl. Add distilled H2O to 1 liter. Dispense the solution into aliquots and sterilize them by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. St
19、ore the buffer at room temperature. TBS containing 0.2% (w/v) sodium azide Triton X-100METHODGrow a 1-liter culture of the appropriate strain of E. coli (e.g., Y1090hsdR, XL1-Blue, or DH1) to stationary phase. Recover the bacteria by centrifugation at 4000g (5000 rpm in a Sorvall GSA rotor) for 20 m
20、inutes at 4C. Pour off the medium, and stand the centrifuge tubes in an inverted position to allow the last traces of medium to drain away. Resuspend the pellet in 100 ml of Cell lysis buffer. Add 200 mg of lysozyme, and incubate the bacterial suspension for 20 minutes at room temperature. Add 1 mg
21、of pancreatic DNase I and 200 l of Triton X-100. Incubate the bacterial suspension for 1 hour at 4C, or until the turbidity clears and the viscosity decreases. Centrifuge the bacterial lysate at 8000g (8200 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4C. Carefully decant the supernatant into a f
22、resh flask. Adjust the pH of the supernatant to 9.0 with 1 M NaOH. Determine the concentration of protein in the lysate using the Lowry, Bradford, or other method of measurement. Chill the extract to 0C, and then bind the bacterial proteins to cyanogen-bromide-activated Sepharose 4B or to Affi-Gel 1
23、0 according to the manufacturers instructions. Before use, equilibrate the Sepharose 4B or Affi-Gel 10 resin containing conjugated E. coli proteins in TBS containing 0.2% (w/v) sodium azide. Use 1 ml of settled volume of resin coupled to E. coli antigen for each milligram of IgG protein to be purifi
24、ed by affinity chromatography. Mix the IgG and the coupled resin and incubate for 12-18 hours at room temperature on a rotating wheel device.Load the slurry into a chromatography column. Recover the antibody by washing the column with TBS. Collect fractions (0.2 column volume each) until the OD280 d
25、rops to zero. Pool the fractions containing antibody, and store the pool at -20C until it is used for immunological screening.REFERENCES1. de Wet, J.R., Fukushima, H., Dewji, N.N., Wilcox, E., OBrien, J.S., and Helinski, D.R. 1984. Chromogenic immunodetection of human serum albumin and alpha-L-fucos
26、idase 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 representations or warranties with respect to the material set forth in this protocol and has no liability in
27、 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 material, please consult:Molecular Cloning: A Laboratory Manual, Third Edition, Joseph Sambrook and David W. Russel
28、l, 2001 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p.14.28-14.30.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OD6001.0，加入IPTG（终浓度1 mM）诱导68 h； 3 收菌，每升菌液约以50 mL PBS重悬，加入1%Triton X-100（v/v），1-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
29、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 加入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电泳检测蛋白纯度，Bradford法检测蛋白浓度； 12 将蛋白置于-20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 fusion protein purification
30、GST融合蛋白纯化1)grow 20ml cells O/N37 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 702)extract cells (from 500ml)in 25 mls.Heintz Buffer plus triton (HBT)by gentle pipette resuspending on ice circa 10after thawing.3)transfer to
31、 50ml conical 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% power,freeze in lN,thaw at37 sonicateagain on ice,solution should become viscous.6)add 1mg DNAse and RNAse,incubate on ice 15.7)spin 7.5
32、k rpm4ss34 10.8)transfer supernatants to conical screw caps,freeze in lN2,may store.9)Batch adsorb w/ 4ml 50% slurry GT-Sepharose (PL 17-0756-01),1hr,4 spin 2,4k on bench.10)aspirate,resuspend in 25ml HBT,spin,repeat.11)pour slurry into column (Econo 1.7x20),elute to top,then with 20 column vols.of
33、HB-T.12)elute protein in minimal volume (5-10ml)HB 5mM GT (Sigma G4251,1.5 mg/ml).13)lN2 freeze as 100ml aliquots for GS.HB 1 literFinal Stock ml/l 25mM HEPES,pH7.9 1M 50 1mM EDTA,pH 8.0 0.5M 2 20% glycerol stock 200 1mM MgCl21M 1 60mM KCl 2M 30 1% Triton stock 10 add before use 0.5mM DTT 1M 0.5 0.5
34、mM PMSF 0.5mM 10 5g/ml Leupeptin 5mg/ml dilute before use 5mg/ml antipain check pH!GUSHISTOCHEMICAL STAINING1)determine number of slides needed,multiply by 0.75ml 2)make up required vol of stain:for 10ml4 5mg X-Gus50l nn dimethyl formamide,dissolve10ml 50mM NaPO4 pH 73)sections best cut with vibrati
35、ng knifefor sections w/ chlorophyll,put in cell-wells w/ 500l stainfor sections w/out chlorophyll,put directly on slides w/ stain4)inc o/n37in humidity chamber5)asp,inc 10in FAA:for 200ml 4 10ml formaldehyde10ml HAc75ml EtOHH2O vol6)inc 2 50% EtOH7)inc 2 100% EtOH8)inc 1 H2OPurification of GST fusio
36、n proteins in E.coli GST融合蛋白纯化，方法一Making GST fusion proteins:(07/19/03)ver.1Grow up 5ml LB with Amp o/n.Add to 45ml LB with Amp37o shake 2.5- 3 hrs,till OD600 0.4-0.8Put bottles in room temperature water for 10 min to cool down.Add 100l 0.2M IPTG to 0.4mM finalShake 30 2hrPellet bacteria,decant sup,
37、invert to drainResuspend in 1ml NETN 0.2mM PMSF / 50ml LBPMSF,stock 10mMNETN: 20mM Tris-Cl (pH8)100mM NaCl1mM EDTA0.5% NP40store at 4Vortex to mix wellSonicate at scale 5 for 15sec.Keep on ice.For 10ml Corning tubes,use scale 7Spin 4,5minTransfer supernatant to a new tube.To each lysate,add 60l 50%
38、Glutathione-Sepharose 4BPepette 400l Sepharose stock (75%)Spin 1000rpm 5min,discard supernantantWash 3x300l NETNResuspend in 300l NETN to get 50% beadsMix in cold room for 2 hours,slowly whirlPellet beads by brief centrifugation,carefully discard supernatantWash 3x1ml NETN/PMSFWash 2x1ml Elution Buf
39、fer (50mM Hepes,pH7.9,40mM KCl,1mM EDTA 1mM DTT)Elute proteins by mix beads with 60l eachElution buffer5mM Glutathione,(for 10mM,use 3.07mg/ml)1mM DTTSlowly swirl at RT 1hrQuick spin to pellet,transfer supernatant to a new tubeRe-elute with 60l eachNETN5mM Glutathione1mM DTTSlowly swirl at RT 30minQ
40、uick spin,combine supernatant,spin and transfer supernatant twice to avoid any residual beads.total is 120l now.Dialyze vs 50% glycerol/10mM Hepes,pH7.5/ 40mM KCl/ 1mM EDTA/ 1mM DTT/ 1mM PMSF in cold room for 2hr or o/n,store at -20Proteins can also be concentrated in a Centriprep-30 concentrator.Th
41、e pore size of the membrane in the Centriprep-30 allows glutathione to pass into the aqueous compartment.PBS can be added to the protein concentrate and the concentration procedure can be repeated.Thinking aliquot and save at -80Run 12% SDS-PAGEPurification of GST fusion proteins in E.coli GST融合蛋白纯化
42、，方法二GST Protein Prep.Ver.21)Grow 50ml of culture in LB or TB antibiotic o/n at 37 shaker.2)Dilute culture in LB or TB antibiotic 1:103)Grow 3hrs at 37.4)Induce culture by adding 0.4 mM IPTG final concentration.(For 50 ml final culture add 20 l of 1M IPTG).5)Grow at 25 for 1 hr.6)Spin 5 min at 5 K7)W
43、ash pellet with half volume of cold H2O.(For 50 ml culture use 25 ml H2O.)8)Wash bacteria again.9)Resuspend in 1 ml of resuspension buffer per 50 ml of culture.Resuspension Buffer- NETN protease inhibitors20 mM Tris pH 8.0100 mM NaCl1 mM EDTA0.5% NP-40 or Triton-X1 g/ml Aproteinin1 g/ml PMSF1 g/ml B
44、enzaminideNote: Tim has 100x stock of protease inhibitors.10)Sonicate for 2x for 10 seconds in cold room.11)Pellet debris by spinning at 4 (Easiest way is to put samples in multiple eppendorfs and spin in cold room at max for 2 min.Binding of Fusion Protein to Beads1)Pipette 400 l of GST-Sepharose i
45、nto Eppendorf2)Spin beads 15 sec 8,000 RPM3)Wash beads 2x with 4 NETN4)Resuspend pellet in 320 l of NETN (Final volume will be 550 l)and put into 4 eppendorfs.5)Add 300 l of resuspension buffer and 75 l of E.Coli.GST Lysate to each tube.6)Incubate for 30 min while rocking in cold room.7)Spin 15 seco
46、nds at 8,000 rpm.8)Wash 3x with 600 l of resuspension buffer9)Remove supernatant and resuspend in appropriate volume resuspension buffer.(You can add this directly to SDS load buffer for running on a gel.)Purification of GST fusion proteins in E.coli GST融合蛋白纯化，方法三，纯化小量Small scale fusion protein prep
47、arationGrow 5ml culture o/n in TB with amp.Add o/n culture to 50ml of TB amp and grow for 3 hours in 37 shaker.Induce culture by adding 20l of 1M IPTG (final 0.4mM)and transfer to 25 shaker for 1hour.Pellet Bacteria 10min at 3KResuspend bacteria in 1ml of NETN protease inhibitors.Sonicate 2x for 5-10seconds each time.Spin out cell debris by spinning in cold microfuge 5 min.at max.Remove supernatant and store at -70.Binding fusion protein lysate to beadsRemove 400l of GST beads into eppendorf and spin 15 sec at 8k.Remove superna