Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes.
Post-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 co-expression 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-1alpha (HIF-1alpha) 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-1alpha 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.
Sugase
Kenji
K
Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Landes
Mindy A
MA
Wright
Peter E
PE
Martinez-Yamout
Maria
M
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats
D001426:Bacterial Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004798:Enzymes; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C034284:IgG Fc-binding protein, Streptococcus; D010455:Peptides