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7100887 
Journal Article 
Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction 
Ding, N; Yang, C; Sun, S; Han, L; Ruan, Yao; Guo, L; Hu, X; Zhang, J; , 
2017 
Yes 
Biochemical and Biophysical Research Communications
ISSN: 0006-291X
EISSN: 1090-2104 
ACADEMIC PRESS INC ELSEVIER SCIENCE 
SAN DIEGO 
138-143 
28188786 
WOS:000396641700023 
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.