重组蛋白质表达复性与纯化.ppt

Type I: chaperones which are functionally responsible for the correct folding, assembly and transport of newly synthesized peptide– HSP family,GroELS,ect. Type II: chaperones with enzymatic properties accountable for acceleration of rate limiting steps of protein folding– PDI（protein disulfide isomerase）, PPI（ peptidyl-prolyl cis-trans isomerase), etc 两种主要的分子伴侣 人PDI的结构特征 PDI 作用机理 PDI has been shown to catalyze reactivation of non-native proteins without disulfide bonds. This function is independent from the redox/isomerase function, as it has been shown to exist in vitro with several model substrates that do not contain disulfides in their native structure. Folding assistant/chaperone Timed addition of PDI to refolding Fab/red. Marcus Mayer et al., BiP and PDI Cooperate in the Oxidative Folding of Antibodies in Vitro, JBC Vol. 275, No. 38, 2000 Marcus Mayer et al., BiP and PDI Cooperate in the Oxidative Folding of Antibodies in Vitro, JBC Vol. 275, No. 38, 2000 PPI的作用 Proline isomerization is widely accepted to be one of the rate-determining steps in protein folding. The function of PPIis to accelerate the cis-trans isomerization process. Molecular Chaperon Cyclophilin Family, 典型的PPI Refolding of HCA II HCA II HCA II with PPl HCA II with PPl and CsA P202N mutant with PPI P202N mutant Reactivation was initiated by rapid dilution of 0.3 M GuHCl and a protein concentration of 0.025 mg/ml (0.83 ,uM). PPI: 9.6μM ; CsA: 25μM 表达量不够高； 包含体在8M尿素中不能溶解； 重组蛋白在大肠杆菌中表达的分子量偏小； 带His-tag重组蛋白不吸附到Ni-chelating树脂上； Ni-chelating分离纯化的效果不好； GST融合蛋白不吸附到glutathione-Sepharose上； 包含体来源的采用透析法或稀释法复性时全部沉淀； Ni-chelating错用DTT后发生黑色沉淀该怎么办？ 贵重的亲和层析树脂经常发生结块该怎么办？ 某些表达载体的表达效率在放大培养时无法提高 工作中经常碰到的问题 尝试不同表达载体， 特别是N端有融合蛋白的载体 是不是忘了加还原剂（DTT或巯基乙醇）？ 是不是在－20℃冻存过？ 用4M盐酸胍试试 是不是酸性蛋白质？ 是不是蛋白质的合成提前中止了？ （富含Arg, Ile, Leu, Pro这几种氨基酸） 可以尝试用BL21 CodonPlus RIL 或 RP 作宿主菌 (Stratagen)； 使用C端带His-tag的融合表达载体（如pET21, Novagen）以便纯化全长的融合蛋白 His-tag被操作过程混入的重金属离子所饱和，可以通过添加0.5 mM EDTA来去除重金属离子的干扰； His-tag被包裹在不易和树脂发生结合的位置（比较