Identification of N-terminal acetylation of recombinant human prothymosin α in Escherichia coli

Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin α is an essential protein in immune stimulation and apoptosis regulation. The protein is N-terminal acetylated in eukaryotes, but similar modification has never been found in recombinant protein produced in prokaryotes. In this study, two mass components of recombinant human prothymosin α expressed in Escherichia coli were identified and separated by RP-HPLC. Mass spectrometry of the two components showed that one of them had a 42 Da mass increment as compared with the theoretical mass of human prothymosin α, which suggested a modification of acetylation. The mass of another one was equal to that of the theoretical one. Peptides mass spectrometry of the modified component showed that the 42-Da mass increment occurred in the N-terminal peptide domain, and MS/MS peptide sequencing of the N-terminal peptide found that the acetylated modification occurred at the N-terminal serine residue. So, part of the recombinant human prothymosin α produced by E. coli was N-terminal acetylated. This finding adds a new clue for the mechanism of acetylated modification in prokaryotes, and also suggested a new method for production of N-terminal modificated prothymosin α and thymosin α1.