Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction

Abstract Escherichia coli 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. coli 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 Pgl B 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. coli cells.