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论文类型：期刊论文

发表时间：2017-03-25

发表刊物：BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

收录刊物：SCIE、PubMed

卷号：485

期号：1

页面范围：138-143

ISSN号：0006-291X

关键字：Glycosylation efficiency; Escherichia coli; Auto-induction; FN3

摘要：Escherichia colt cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. cob is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto -induction procedures. To justify the potential for general application of the optimized auto -induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. colt cells. (C) 2017 Elsevier Inc. All rights reserved.