Wu Jun, Chang Shaohong, Gong Xing, Liu Dianxin, Ma Qingjun
Beijing Institute of Biotechnology, Fengtai District, Beijing 100071, China.
Biochim Biophys Acta. 2006 Aug;1760(8):1241-7. doi: 10.1016/j.bbagen.2006.04.001. Epub 2006 Apr 19.
Functional modification of protein through N-terminal acetylation is common in eukaryotes but rare in prokaryotes. Prothymosin alpha 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 alpha 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 alpha, 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 alpha 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 alpha and thymosin alpha1.
蛋白质通过N端乙酰化进行功能修饰在真核生物中很常见，但在原核生物中很少见。原胸腺素α是免疫刺激和细胞凋亡调节中的一种重要蛋白质。该蛋白质在真核生物中进行N端乙酰化，但在原核生物中产生的重组蛋白中从未发现过类似修饰。在本研究中，通过反相高效液相色谱法（RP-HPLC）鉴定并分离了在大肠杆菌中表达的重组人原胸腺素α的两个质谱成分。对这两个成分进行质谱分析表明，其中一个成分与人类原胸腺素α的理论质量相比有42 Da的质量增加，这表明存在乙酰化修饰。另一个成分的质量与理论质量相等。对修饰成分进行肽质谱分析表明，42 Da的质量增加发生在N端肽结构域，对N端肽进行串联质谱（MS/MS）肽测序发现乙酰化修饰发生在N端丝氨酸残基上。因此，大肠杆菌产生的部分重组人原胸腺素α进行了N端乙酰化。这一发现为原核生物中乙酰化修饰的机制增添了新线索，也为生产N端修饰的原胸腺素α和胸腺素α1提出了一种新方法。
