GST蛋白表达、纯化步骤.doc

Protein Expression Protocol:1. Transform control construct and tag-fusion-constructs into E.coli BL21(DE3) competent cell, grow overnight; 2. Inoculate single colony to 23 ml LB medium, and grow at 37 degree for 710 hours or overnight;3. 1:100 inoculate to 3 ml LB, bacteria grow for 2-3 hours at 37 degree, then follow 1:2000 add 1 M IPTG to induce protein expression overnight at 22 degree;4. Spin down bacteria at 12000 rpm for 1 min, wash with 1 ml ddH2O, add 100 ul 1x PBS to resuspend the deposition, then sonicate 35 min on ice;5. Spin down at 12000 rpm for 10min, separate the supernatants and deposition;6. Boiling with loading buffer at 100 degree for 5min, Spin down at 12000 rpm for 5min, 12% SDS-PAGE;7. After confirmed protein expressed in supernatants, increase the volume of bacteria medium. 1:100 inoculate to 100 ml LB, grow bacteria for 2-3 hours at 37 degree; then follow 1:2000 add 1 M IPTG to induce protein expression overnight at 22 degree;8. Spin down bacteria at 8000 rpm 4 degree for 5 min, wash with 10 ml ddH2O, add 5 ml 1x PBS to resuspend the deposition, then sonicate 60 min on ice;9. Spin down at 12000 rpm 4 degree for 10min, separate the supernatants and deposition;10. Prepare the supernatants for purification.蛋白表达注意事项：1. E.coli BL21比DH5生长要快，固体培养基上1012小时即可长斑，液体培养基中8小时后即可生长成较大密度；切勿培养时间过长，防止菌体老化；2. 为了后续实验的便利，应尽量减少包涵体的形成。主要有以下方法可以减少包涵体的形成:a. 降低IPTG的浓度。IPTG终浓度0.20.8M都可以诱导细菌表达蛋白；b. 加入IPTG后，把细菌生长温度降至1630度。较低的生长温度降低了无活性聚集体形成的速率和疏水相互作用，从而可减少包涵体的形成；c. 添加可促进重组蛋白质可溶性表达的添加剂，培养E.coli时添加高浓度的多醇类、蔗糖或非代谢糖可以阻止分泌到周质的蛋白质聚集反应，在最适浓度范围内添加这些添加剂不会影响细胞的生长、蛋白质的合成或运输，其它添加剂还有乙醇（诱导热休克蛋白的表达）、低分子量的巯基或二硫化合物（影响细胞周质的还原态，从而影响二硫键的形成）和NaCl；d. 供给丰富的培养基，优化培养条件，如供氧、pH等。3. 超声后加入终浓度1% Triton x-100处理3060min，有利于去除膜碎片和膜蛋白；4. 若需表达的蛋白含稀有密码子较多，尝试更换E.coli宿主菌株，如Rosseta。5. 若表达毒性蛋白，造成细菌死亡或者生长缓慢，可以使用pLysS 菌株。GST fusion protein purification（Glutathione Sepharose 4B）Buffer preparation：Water and chemicals used for buffer preparation should be of high purity. We recommend filtering the buffers by passing them through a 0.45 m filter before use.Binding buffer: PBS, pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3)Elution buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0GST-x Purification Protocol1. Determine the volume of Glutathione Sepharose 4B required for your purification. As the bind capacity of Glutathione Sepharose 4B is 5 mg glutathione-S-transferase/ml medium, generally, 150 ul 200 ul slurry for 100 ml LB bacteria lysate is enough; 2. Gently shake the bottle of Glutathione Sepharose 4B to resuspend the slurry;3. Sediment the medium by centrifugation at 12 000 g for 1 min. Carefully decant the supernatant;4. Wash the Glutathione Sepharose 4B by adding 5 slurry volumes Binding buffer. Invert to mix;5. Sediment the medium by centrifugation at 12 000 g for 1 min. Carefully decant the supernatant;6. Repeat steps 3 and 4 twice;7. Trannsfer the medium to a 15 ml tube, add the cell lysate to the prepared Glutathione Sepharose 4B and incubate overnight at 4. Use gentle agitation such as end-over-end rotation;8. Sediment the medium by centrifugation at 6000 g for 5 min. Carefully decant the supernatant and save it for measuring the binding efficiency to the medium i.e. by SDS-PAGE;9. Wash the Glutathione Sepharose 4B by adding 5 slurry volumes Binding buffer, transfer the medium to a 1.5 ml tube. Invert to mix;10. Sediment the medium by centrifugation at 12000 g for 1 min. Carefully decant the supernatant (= wash) and save it for SDS-PAGE analysis;11. Repeat steps 9 and 10 twice for a total of three washes;12. Elute the bound protein by adding 0.5 slurry volume Elution buffer. Incubate at room temperature for 1 h. Using gentle agitation such as end-over-end rotation;13. Sediment the medium by centrifugation at 12000 g for 1 min. Carefully decant the supernatant (= eluted protein);14. Repeat steps 7 and 8 twice for a total of three elutions. Check the three eluates separately for purified protein and pool according to the results;15. Wash the Glutathione Sepharose 4B by adding 5 slurry volumes Binding Buffer. Invert to mix; 16. Sediment the medium by centrifugation at 12000 g for 1 min. Carefully decant the supernatant;17. repeat steps 14 and 15 twice;18. Store Glutathone Sepharose 4B at 4C in 20% ethanol.GST融合蛋白纯化注意事项：1. Binding Buffer和Elution Buffer中加入110 mM DTT，可以提高蛋白纯度，但是会导致其产率降低；2. GST融合蛋白不与Glutathione Sepharose 4B(简称4B)结合：a. 超声太剧烈或时间过长会引起蛋白变性，导致蛋白不能与4B结合。需注意设置好超声仪的功率和间隔时间；b. 在细胞裂解和加Binding Buffer和Elution Buffer之前，加入终浓度110 mM DTT可以显著提高GST融合蛋白的结合效率；c. 由于4B在pH值低于6.5或高于8.0结合效率会降低，因此使用前需用pH6.58.0的Buffer如PBS进行平衡；d. 如果4B已经使用过几次，有必要进行重生或者改用新鲜的4B；3. GST融合蛋白不能有效洗脱：a. 洗脱时间不足；b. 增大Elution Buffer的洗脱体积；c. 加大Elution Buffer中还原型glutathione的浓度。Glutathione的推荐浓度是10 mM，若此浓度洗脱效果不是很理想可以尝试50 mM Tris- HCl, 2040 mM reduced glutathione, pH 8.0 的Elution Buffer；d. 增大Elution Buffer的pH值。Elution Buffer的pH值调至pH 89可以提高洗脱效率而不需要增加glutathione的浓度；e. 增加Elution Buffer的离子强度。加入0.10.2 M NaCl能提高洗脱效率；f. 改用新鲜配置的Elution Buffer；g. Elution Buffer中加入非离子型变性剂。非特异性的疏水作用可能会阻碍GST融合蛋白从4B上增溶和洗脱。加入0.1% Triton X-100 or 2% 和N-octylglucoside可以显著增加洗脱效率。4. Glutathione Sepharose 4