Han Cuifang, Shan Hui, Bi Chuanlin, Zhang Xinling, Qi Jieqiong, Zhang Boyuan, Gu Yuchao, Yu Wengong
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.
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
J Biochem. 2015 Jun;157(6):477-84. doi: 10.1093/jb/mvv006. Epub 2015 Jan 24.
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.
