Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes

Abstract

SynopsisPost-translational modification plays crucial roles in signal transduction in eukaryotic cells. To elucidate the biological function of a protein with a specific post-translational modification, it is necessary to isolate the modified protein. However, it is difficult to incorporate a modified amino acid into a specific position of a protein, in particular, in a large-scale preparation. In order to prepare post-translationally modified proteins in Escherichia coli (E. coli), we have constructed coexpression vectors that contain protein and corresponding enzyme genes. The protein and enzyme are co-expressed in the same E. coli cells and the protein is post-translationally modified in vivo. By using this system, the transcriptional activator cyclic-AMP-response-element-binding protein (CREB) was phosphorylated at Ser-133 and the hypoxia-inducible factor-1α (HIF-1α) was hydroxylated at Asn-803 in E. coli. Although the constructs of the proteins we used are very flexible and susceptible to degradation by proteases in E. coli when they are expressed alone, the B1 domain of streptococcal protein G (GB1) fused to the N-terminus of the proteins increased the yields dramatically. Site-specific phosphorylation of CREB and hydroxylation of HIF-1α were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and NMR. Our GB1-fusion co-expression system can be used in the same way as conventional protein expression in E. coli, making it a flexible and economical method to produce a large amount of a post-translationally modified protein. Keywordsphosphorylation; hydroxylation; co-expression; Escherichia coli; B1 domain of protein G (GB1); CREB; HIF-1α INTRODUCTIONPost-translational modification plays a crucial role in signal transduction in eukaryotic cells by introducing diversity and versatility into proteins translated from genomic DNA sequences [1]. Post-translational modification extends the range of function of the proteins by attaching other biochemical functional groups such as phosphate, hydroxyl group, acetate, lipids, or carbohydrates, causing changes in intrinsic activities of the proteins such as activation, inactivation or change in other properties such as conformational change. Despite the *To whom correspondence should be addressed (email yamout@scripps.edu). † Current address: Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. importance of post-translational modifications in biology, the production of large quantities of mod...

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