title: 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 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. coli cells.
authors.affiliation: School of Life Science and Medicine, Dalian University of Technology, Liaoning 124000, China; Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Ding
authors.affiliation: Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China.
authors.forename: Chunguang
authors.initials: C
authors.lastname: Yang
authors.affiliation: Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China.
authors.forename: Shenxia
authors.initials: S
authors.lastname: Sun
authors.affiliation: Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China.
authors.forename: Lichi
authors.initials: L
authors.lastname: Han
authors.affiliation: Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Ruan
authors.affiliation: Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China.
authors.forename: Longhua
authors.initials: L
authors.lastname: Guo
authors.affiliation: Academic Centre for Medical Research, Medical College, Dalian University, Liaoning 116622, China. Electronic address: huxuejun@dlu.edu.cn.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Hu
authors.affiliation: School of Life Science and Medicine, Dalian University of Technology, Liaoning 124000, China. Electronic address: jnzhang@dlut.edu.cn.
authors.forename: Jianing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D016123:Campylobacter jejuni / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D056426:Genetic Loci; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007218:Industrial Microbiology; D008958:Models, Molecular; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D005353:Fibronectins; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins
keywords: Auto-induction; Escherichia coli; FN3; Glycosylation efficiency