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.