Hewinson R G, Russell W P
Molecular Genetics Unit, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, UK.
J Gen Microbiol. 1993 Jun;139 Pt 6:1253-9. doi: 10.1099/00221287-139-6-1253.
The gene encoding an immunodominant secreted antigen, MPB70, of Mycobacterium bovis was cloned into the plasmid vector pBluescript II KS+ along with its native ribosome-binding site. In this construct translation of the protein in Escherichia coli was from the native AUG initiation codon and was directed by the mycobacterial ribosome-binding site. Two different molecular mass forms (26 kDa and 22 kDa) of MPB70 were observed in whole-cell pellets of recombinant E. coli. The difference in size indicates cleavage of the signal peptide of MPB70 by an endopeptidase of E. coli. MPB70 was secreted into the periplasm of recombinant E. coli, where the 22 kDa form of the protein was predominant. The culture filtrate contained only the 22 kDa form of the protein, which was soluble. The passage of MPB70 from the periplasm into the growth medium was found to be due, at least in part, to non-specific leakage of periplasmic proteins across the outer membrane associated with the expression of recombinant MPB70.
编码牛分枝杆菌一种免疫显性分泌抗原MPB70的基因，与其天然核糖体结合位点一起被克隆到质粒载体pBluescript II KS+中。在这个构建体中，该蛋白在大肠杆菌中的翻译起始于天然的AUG起始密码子，并由分枝杆菌核糖体结合位点指导。在重组大肠杆菌的全细胞沉淀中观察到两种不同分子量形式（26 kDa和22 kDa）的MPB70。大小差异表明MPB70的信号肽被大肠杆菌的一种内肽酶切割。MPB70被分泌到重组大肠杆菌的周质中，其中以22 kDa形式的蛋白为主。培养滤液中仅含有可溶的22 kDa形式的蛋白。发现MPB70从周质进入生长培养基至少部分是由于周质蛋白通过与重组MPB70表达相关的外膜发生非特异性渗漏。
