Lassen S F, Mortensen K K, Sperling-Petersen H U
Department of Chemistry, Aarhus University, Denmark.
Biochem Int. 1992 Aug;27(4):601-11.
A serious problem during purification of the E. coli initiation factor IF2 is a significant loss of native IF2 due to partial degradation. We have previously shown that the major fragment, IF2 gamma (65 kDa), is the result of cleavage of IF2 alpha at the peptide bond between lysine 289 and arginine 290. In this paper we demonstrate that the cleavage is a result of proteolysis by outer membrane protease OmpT occurring immediately after cell lysis and in the S30 supernatant. By protein engineering we have constructed an IF2 mutant Lys289-greater than Met and shown that the IF2 gamma cleavage site in this mutant protein is insensitive to cleavage by OmpT. However the mutant protein is cleaved by OmpT between arginine 279 and alanine 280, which is a novel sequence specificity for this protease.
在大肠杆菌起始因子IF2的纯化过程中，一个严重的问题是由于部分降解导致天然IF2大量损失。我们之前已经表明，主要片段IF2γ（65 kDa）是IF2α在赖氨酸289和精氨酸290之间的肽键处裂解的结果。在本文中，我们证明这种裂解是细胞裂解后立即在外膜蛋白酶OmpT作用下发生的蛋白水解作用的结果，且发生在S30上清液中。通过蛋白质工程，我们构建了一个IF2突变体Lys289→Met，并表明该突变蛋白中的IF2γ裂解位点对OmpT的裂解不敏感。然而，该突变蛋白在精氨酸279和丙氨酸280之间被OmpT裂解，这是该蛋白酶的一种新的序列特异性。
