title: A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E. coli.
abstract: O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-GlcNAcylated protein production in E. coli. The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β-d-thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O-GlcNAcylation levels by altering the inducer concentration. More importantly, the O-GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O-GlcNAcylated proteins in E. coli.
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Cuifang
authors.initials: C
authors.lastname: Han
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Shan
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Chuanlin
authors.initials: C
authors.lastname: Bi
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Xinling
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Jieqiong
authors.initials: J
authors.lastname: Qi
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Boyuan
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China guych@126.com.
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao 266003, China yuwg66@ouc.edu.cn.
authors.forename: Wengong
authors.initials: W
authors.lastname: Yu
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001089:Arabinose; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D000117:Acetylglucosamine
keywords: CKII; O-GlcNAc; OGT; coexpression; p53