Efficient production of secretory Streptomyces clavuligerus ?-lactamase inhibitory protein (BLIP) in Pichia pastoris.

ABSTRACT: ?-Lactamase inhibitory protein (BLIP), a low molecular weight protein from Streptomyces clavuligerus, has a wide range of potential applications in the fields of biotechnology and pharmaceutical industry because of its tight interaction with and potent inhibition on clinically important class A ?-lactamases. To meet the demands for considerable amount of highly pure BLIP, this study aimed at developing an efficient expression system in eukaryotic Pichia pastoris (a methylotrophic yeast) for production of BLIP. With methanol induction, recombinant BLIP was overexpressed in P. pastoris X-33 and secreted into the culture medium. A high yield of ~?300 mg/L culture secretory BLIP recovered from the culture supernatant without purification was found to be >?90% purity. The recombinant BLIP was fully active and showed an inhibition constant (Ki) for TEM-1 ?-lactamase (0.55?±?0.07 nM) comparable to that of the native S. clavuligerus-expressed BLIP (0.5 nM). Yeast-produced BLIP in combination with ampicillin effectively inhibited the growth of ?-lactamase-producing Gram-positive Bacillus. Our approach of expressing secretory BLIP in P. pastoris gave 71- to 1200-fold more BLIP with high purity than the other conventional methods, allowing efficient production of large amount of highly pure BLIP, which merits fundamental science studies, drug development and biotechnological applications.

SUBMITTER: Law KH 

PROVIDER: S-EPMC5910447 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature