GST蛋白表达、纯化步骤.docVIP

Protein Expression Protocol: Transform control construct and tag-fusion-constructs into E.coli BL21(DE3) competent cell, grow overnight; Inoculate single colony to 2~3 ml LB medium, and grow at 37 degree for 7~10 hours or overnight; 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; 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 3~5 min on ice; Spin down at 12000 rpm for 10min, separate the supernatants and deposition; Boiling with loading buffer at 100 degree for 5min, Spin down at 12000 rpm for 5min, 12% SDS; 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; 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; Spin down at 12000 rpm 4 degree for 10min, separate the supernatants and deposition; Prepare the supernatants for purification. 蛋白表达注意事项： BL21比DH5α生长要快，固体培养基上10～12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； 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: 降低IPTG的浓度。IPTG终浓度0.2～0.8μM都可以诱导细菌表达蛋白； 加入IPTG后，把细菌生长温度降至16～30度。较低的生长温度降低了无活性体形成的速率和疏水相互作用，从而可减少包涵体的形成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供给丰富的培养基，培养条件，如供氧、pH等。E.coli宿主菌株，如Rosseta。 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 μm filter before use.Binding buffer: PBS, pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 m