期刊
JOURNAL OF BIOTECHNOLOGY
卷 168, 期 4, 页码 315-323出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2013.10.008
关键词
O-GlcNAc; beta-N-Acetylglucosaminidase; Glycosidase; E. coli; NagZ
资金
- Louisiana State University HHMI Summer Undergraduate Research Program
- Louisiana State University
The O-linked beta-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding beta-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli beta-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering beta-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-beta 1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-beta-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system. (C) 2013 Elsevier B.V. All rights reserved.
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