含绿脓杆菌外毒素A受体结合区重组蛋白的纯化

Purification of Recombinant Protein Containing ReceptorBinding Domain of Exotoxin A of P aeruginosa

将表达含绿脓杆菌外毒素（ＰＥＡ）受体结合区基因的质粒ｐＥＴ－ＥＡＢ转化到大肠杆菌ＢＬ２１（ＤＥ３）中，经ＩＰＴＧ诱导表达了含ＰＥＡ受体结合区的重组蛋白（称ＰＥ３４）。ＰＥ３４主要以包涵体形式存在于大肠杆菌中，用溶菌酶－脱氧胆酸钠法结合超声波裂解法破碎细菌，离心制备包涵体。用２ｍｏｌ／Ｌ脲洗涤包涵体后，包涵体纯度可达７５％，在８ｍｏｌ／Ｌ脲存在条件下，ＳｅｐｈａｃｒｙｌＳ－２００凝胶滤过，ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ离子交换层析纯化，获得ＳＤＳ－ＰＡＧＥ１条带的ＰＥ３４，纯度达９５．８％，得率为２４．５％。

Expressing plasmid containing receptorbinding domain of exotoxin A ofP aeruginosa (PEA), pETEAB, was transformed into E coli BL21(DE3). By inducing of IPTG, a recombinant protein containing receptorbinding domain of PEA, named PE34, was expressed. PE34 formed inclusion body in expressed E coli. The expressed E coli was splitted by lysozymedeoxycholic acid sodium and supersonic. The inclusion body was prepared by centrifugation and washing with 2 mol/L urea with the purification rate of the inclusion body being above 75%. Then it was dissolved by 8 mol/L urea and further purified by Sephacryl S200 gel filter and DEAESepharose F F ionexchange chromatography. the purified PE34 appeared a single band in SDSPAGE gel with its purification rate and recovery rate being 95.8% and 24.5%.