耐冷希瓦氏菌外膜蛋白的体外折叠研究

目的:旨在建立耐低温革兰氏阴性菌外膜蛋白体外折叠体系,为膜蛋白合成耐低温机制提供理论基础。方法:以包涵体的形式在大肠杆菌中过量表达了来源于耐低温希瓦氏菌的OmpA同源外膜蛋白Omp74的全蛋白质和N端跨膜结构域,纯化包涵体后,用高浓度尿素或强阴离子表面活性剂溶液溶解包涵体,以非离子表面活性剂为折叠介质,建立该外膜蛋白的体外折叠体系,同时以大肠杆菌的OmpA作为对照进行了比较研究。结果:与OmpA相比,Omp74体外折叠受温度影响较小,低浓度的阴离子表面活性剂能促Omp74的折叠,但对OmpA的折叠没有影响;C端结构域抑制Omp74在表面活性剂中的折叠;Omp74在0.5%的月桂酰基麦芽糖苷(DDM)和0.4%的十二烷基肌氨酸钠的混合溶液中能达到接近100%的折叠效率。

In vitro Refolding of an Outer Membrane Protein from a Psychrotrophic Shewanella sp.

Objective: To investigate cold-adapatation mechanisms of outer membrane protein folding of psychrotrophic Gram-negative bacterium by constructing the in vitro refolding system. Methods: The outer membrane protein Omp74 from a psychrotrophic bacterial Shewanella sp. was overexpressed as inclusion body in E. coli, purified, dissolved in surfactant or high concentrations of urea, and refolded with non-ionic surfactant. OmpA from E. coli was used as a control from mesotrophic bacteria. Results: Being different from OmpA, the refolding of Omp74 was not sensitive to temperature, and low concentration of sarkosyl facilitated the folding of wild type Omp74 but showed no effect on OmpA folding; C-terminal periplasmic domain inhibited the folding; complete refolding this recombinant OMP in vitro in 1%DDM mixed with 0.4% sarkosyl.