Expression, purification and identification of GST fusion protein

1、Expression, purification and identification of GST fusion protein Protein Purification by Affinity Chromatography Protein Separation by SDS-PAGE Specific Protein Identification by Western-blotting Contents1. Induced Expression of GST fusion protein in E coli. P1142. Protein Separation of by SDS-PAGE

2、. P383. Protein Identification by Western-blotting. P114工程菌中外源GST融合蛋白的分离纯化及鉴定1、E coli菌中外源GST融合蛋白的诱导表达(IPTG诱导)2、特异外源GST融合蛋白表达的鉴定（Western-blotting）3、细菌的裂解（溶菌酶法 lysozyme）4、 Glu-Agarose beads亲和层析分离GST fusion蛋白5、 SDS-PAGE鉴定GST fusion蛋白的纯度Determining Protein Expression by Western-blottingGST (Glutathione

3、S-transferases) protein expression in E. coliWestern blotting ( Immunoblotting )1. DefinitionA technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. So called since it has some similarity to a Southern blotting.2. Why we have to use it?To identify a tar

4、get protein in a complex mixture, and measure its expression level.Pre-Procedure of Western blotting Protein extraction: Sample come from cells, serum/plasma, tissues and culture supernatant Measurement of Protein concentration (For Western Blotting: 20-60 mg/well) Steps of Western blottingStep1. Us

5、e gel electrophoresis to separate proteins PAGE ( polyacryamide gel electrophoresis ) or SDS-PAGE - Sodium dodecyl sulfate PAGE.Step2. Transfer the proteins in the gel onto a NC (nitrocellulose) or PVDF membrane Step3. Staining: use the specific antibody to identify the target proteinProcedure of We

6、stern blotting1. Gel preparation2. loading Sample： Sample (50100 g/well ) + equal volume of 2×SDS-PAGE loading buffer，100 for 35 min，loading Sample and prot. Marker.3. Running the gel: Apply 8V/cm gel until the proteins are well into the stacking gel, then 15V/cm gel until the tracking dye reac

7、hes the bottom of the gel.Membrane transfer: soak the NC membrane and filter papers in transfer buffer before assembling the transfer apparatus. Assemble the transfer apparatus according to the following sequence. Avoid air bubbles between any of the layers .4°C，60mA, overnight.Blocking membran

8、e: block the membrane in 1015 ml 5% BSA in TBS-T for 1 hour of (rotation)Primary antibody incubation: Remove the blocking solution and incubate the membrane with 10 ml primary antibody for 2 hours at room temperature(rotation) or overnight at 4°CWash the membrane: Remove the antibody and perfor

9、m 3 room temperature washes (15 minutes of rotation each) with 30ml TBS-T.Secondary antibody incubation: Incubate the membrane with 10ml secondary antibody for 1 hour at room temperature (rotation)Wash the membrane: emove the antibody and perform 3 room temperature washes (15 minutes of rotation eac

10、h) with 30ml TBS-T.Developing: (Use of chromomeric substrate- 3,3-diaminobenzidine, DAB) DAB is most sensitive for immunocoupled horseradish peroxidase. It is converted in situ a brown precipitate. -Transfer the membrane to a shallow tray.Procedure of Western blottingTransfer the membrane to a shall

11、ow tray.-Add 10ul H2O2 (30%) to 10 ml of 0.05%DAB in PBS, mix well immediately;-Pour the DAB to the membrane, incubate at room temperature with gentle shaking in the dark if possible.-Monitor the progress of the reaction carefully. When the bands are of the desired intensity (2-10min), wash the filt

12、er briefly in water, and in PBS. -Dry the membrane and photograph it to provide a permanent record of the experiment.Western blottingStripping Membranes: * Put the membrane in PBS or TBS with 7ul/ml -mercaptoethanol and 2%SDS for 30' at Room temperature in agitation.* Wash the membrane for

13、30' in PBS or TBS. * Put the membrane in blocking buffer注意：硝酸纤维素(NC)膜是通过疏水作用和蛋白质相联，反复洗几次后，蛋白易掉下来，结果较差。尼龙膜主要通过它膜上的正电荷和蛋白接合（常用的PVDF即带正电荷的尼龙膜），同时也有疏水作用，这样蛋白在PVDF膜上接合较牢，不易脱落，结果较好。Western blotting troubleshooting* No target protein？1. No expression；2.Target protein Degraded；3. Antibody does not recog

14、nize the target protein* Precipitation in cell extracts1. Incomplete protein denaturation: Increase the amount of SDS and the boiling time of sample；2. Too much antigen: Add more sample buffer* Target protein band very weak？ Increase the loading of sample. Decrease the dilution times of primary anti

15、body.* The molecular weight of the antigen is doubled? Dimer of the antigen is formed. Add more DTT and elongate the boiling time.* Too much background !Dcrease the loading of sample and the primary concentration, adjust the primary antibody incubation time，increase the blocking time.Special reagent

16、Transfer buffer: 10% methanol, 24 mM Tris, 194 mM glycine (Caution: The buffer must be at 4 for electrophoretic transfer.)Washing buffer (TBS-T): 100 ml 10X TBS + 900 ml dd H2O + 1 ml Tween-20Staining solution： TBS-T-10 ml; DAB-6 mg; 30% H2O2Purification of GST fusion protein by Affinity chromatogra

17、phy GST (Glutathione S-transferases) protein purification by Glu-Agarose beadsGST酶的亲和层析原理* 谷胱甘肽(Glutathione，Glu)与谷胱甘肽巯基转移酶( Glu S-Transferase )之间具有特异性的作用力。将混合菌体蛋白与谷胱甘肽琼脂糖凝胶珠（Glu-Agarose beads）孵育，凝胶手臂上的Glu可以与GST蛋白特异性结合，通过洗脱除去不能与珠结合的杂蛋白而获得琼脂糖珠结合的GST纯蛋白。* 进一步使用还原型谷胱甘肽(GSH)洗脱时，将竞争GST上的结合位点而将GST融合蛋白洗脱下来，

18、从而获得纯化的GST融合蛋白的纯品。1.在含细菌沉淀的Ep管中加入500 l 的裂解液悬浮细菌，再加入 50 l 溶菌酶溶液，反复在手中颠倒振摇30 min，直至管内液体变得清亮，10000g离心5min后收集上清。（留存10l上清于一新Ep管中做对照）2.将收集的剩余上清直接放入含有50l 50% Glu-Aragrose beads的EP管中，混匀管内液体，振摇反应5min后，2000g离心1min，然后小心去除珠子上方的上清溶液。3.在Ep管中加入预冷的PBS洗液1ml悬浮珠子，2000g离心1min，小心去除上清溶液，重复用PBS洗珠子8次以上, 最后一次用5000g 离心2min。最

19、后将珠子上方的溶液尽量吸干净，注意操作中尽量避免珠子被溶液带走。4.在收集的珠子中加入20l GSH溶液悬浮珠子（注意不要颠倒Ep管，以免珠子粘在管壁上），室温反应5min后，5000g离心1min后收集上清溶液（即纯化的GST蛋白）至一新Ep管中。（回收含有珠子的Ep管）蛋白质的凝胶电泳分离Protein gel electrophoresis琼脂糖凝胶电泳 （Agarose Electrophoresis ）聚丙烯酰胺凝胶电泳(Polyacrylamide gel electrophoresis, PAGE）常规PAGE（Native PAGE）不连续PAGE (Discontinuous

20、 PAGE)十二烷基磺酸钠聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate PAGE ， SDS-PAGE)固相pH梯度等电聚焦 (Immobilised pH gradient isoelectric focusing, IPG-IEF)双向电泳( Two-dimensional electrophoresis, 2-DE)Advantages of PAGE* Polyacrylamide gel is a synthetic polymer and a gel matrix with different pore size and sieving effect.

21、 * Good reproducibility* Good mechanical strength and elasticity, easy to handle.* No electroosmosis (电渗作用)* High Resolution* Widely used for isolation and identification of proteins, nucleic acids and peptidesPrinciple of gel polymerizationPolyacrylamide: Acrylamide丙烯酰胺which forms a linear polymer,

22、 can be cross-linked with N,N-methylene bisacrylamide N,N-亚甲基双丙烯酰胺, to form a gel matrix of controlled pore size.Polymerization is catalyzed by free radicals, generated by ammonium persulfate (AP，过硫酸铵) in the presence of Tetramethylethylenediamine (TEMED,甲基乙二胺).Discontinuous gel systemThe Stacking G

23、el 浓缩胶: * Approx. 10% of the volume of the total gel and a lower %(2.5-4.5%) acrylamide and a lower pH (6.8). * Charged molecules move freely and proteins in a sample should accumulate in stacks of closely spaced bands before encountering the separating gel. * An ion gradient : chloride ions >pro

24、teins > glycinate ionsThe Separating gel 分离胶: * A higher % (7-15%) acrylamide and at a higher pH(8.8) * Proteins are separated into discrete bands based on size. * The molecular weights of protein can be estimated by measuring the mobility of protein standards on the same gel. SDS-PAGESDS (sodium

25、 dodecyl sulfate, 十二烷基磺酸钠)An anionic detergentDenatures native proteins to individual polypeptides.* SDS binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide.* SDS coats polypeptides with an approximately uniform charge-to-mass ratio of (-) charge, and polypeptides are approxim

26、ately uniformly shaped into spheres.* Mobilities of these polypeptides will be a linear function of the logarithms of their molecular weights logMW=K-bRf, (MW为分子量，Rf为relative mobility相对迁移率，k、b均为常数)* Separation on SDS-PAGE is not on the basis of shape or intrinsic charge of the protein, the exact rat

27、e of movement of a particular protein depends on its size. * Dithiothreitol(DTT) 二硫苏糖醇 or 2-mercaptoethanol 巯基乙醇 denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). Man

28、y insoluble proteins are then dissolves and combined with SDS.* Molecular weight of some proteins cannot be determined by SDS-PAGE: proteins with abnormal charge or conformation, proteins with big prosthetic groups (some glycoprotein）structural proteins (collagen), etc. Principle of SDS-PAGE* Separa

29、tion on SDS-PAGE : the exact rate of movement of a particular protein depends on its size. * SDS coats proteins with an approximately uniform charge-to-mass ratio of (-) charge , and proteins are approximately uniformly shaped into spheres, thus, proteins separated by SDS-PAGE are denatured. * Disul

30、fides linkages in proteins are broken by addition of ß-mercaptoethanol(ß-巯基乙醇) or 1, 4-dithiothreitol (DTT二硫苏糖醇 ), thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits),so running proteins in SDS-PAGE are reduced conditio

31、n. Many insoluble proteins are then dissolves and combined with SDS.缓冲液系统 Buffer systems浓缩胶缓冲液：0.5 mol/L Tris-Cl, pH6.8分离胶缓冲液：1.5 mol/L Tris-Cl, pH8.8电泳缓冲液(pH8.3)：0.025 mol/L Tris 0.2 mol/L Gly(甘氨酸)样品上样缓冲液：2 X SDS-PAGE Loading buffer100 mmol/L Tris-Cl (pH 6.8)200 mmol/L 二硫苏糖醇(DTT)4% SDS0.2% 溴酚蓝 Brom

32、ophenol blue20% 甘油 Glycerol Procedure1. Gel Cassette Assembly (1) Clean and completely dry pertinent materials(2) Set up your gel apparatus2. Preparation of the Gel 10% Separating gel 5% Stacking Gel Acrylamide is a neurotoxin. Dont discard unpolymerized acrylamide gel solution into the sink. 配胶:先配分

33、离胶，再配浓缩胶。(在孵箱里中聚合)* 10%分离胶配方：（10ml,一块胶）ddH2O 3.90 ml 30%凝胶贮液 3.33 ml分离胶buffer 2.50 ml 10% SDS 100 l10% AP 100 l10%TEMED 100 lAllow to polymerize by overlaying gently with water.* 4.8%浓缩胶配方：（5ml）ddH2O 3.345 ml30%凝胶贮液 0.8 ml浓缩胶buffer 0.625 ml10%SDS 50 l10% AP 50 l10%TEMED 50 ldecant the overlay, pour

34、the stacking gel, Insert the comb and allow to polymerize.(2) Once the gel has polymerized, the comb can be gently removed.3. Sample preparation(1) Combine 20 l of sample (a unknown protein) with 20l of 2X loading buffer (1:1);20-100g protein per well (Prot. Conc. was previously determined)(2) Heat samples for