Babbitt P C, West B L, Buechter D D, Kuntz I D, Kenyon G L
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Biotechnology (N Y). 1990 Oct;8(10):945-9. doi: 10.1038/nbt1090-945.
Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity. Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein. We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding. While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation. This treatment improves the recovery of active CK approximately 100-fold. We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E. coli. Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well.
将来自加州电鳐电器官cDNA的肌酸激酶（CK）在大肠杆菌中表达，会产生一种不溶性蛋白质产物，且未检测到CK活性。尽管这是一种稳定的聚集体，可通过离心以富集形式分离出来，但最初通过变性和复性来产生酶活性的尝试仅产生了微量的活性蛋白。我们发现，这些低回收率是由于CK在变性和复性过程中发生了蛋白水解。虽然这种蛋白水解活性不受苯甲基磺酰氟（PMSF）或乙二胺四乙酸（EDTA）的抑制，但在变性之前，通过用含去污剂的缓冲液萃取，可以从CK聚集体中大量去除这种活性。这种处理使活性CK的回收率提高了约100倍。我们还发现，当牛胰蛋白酶抑制剂（BPTI）的突变体在大肠杆菌中表达时，形成的聚集体也具有类似的蛋白水解活性。在两种不同的表达系统中发现这种蛋白水解活性表明，对于从其他包涵体中回收活性蛋白而言，也应将其视为一个潜在问题。
