Fang Hongqing, Zhang Xu, Shen Lin, Si Xinxi, Ren Yuantao, Dai Hongmei, Li Shulong, Zhou Changlin, Chen Huipeng
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, People's Republic of China.
Appl Microbiol Biotechnol. 2009 Aug;84(1):99-104. doi: 10.1007/s00253-009-1994-8. Epub 2009 Apr 8.
N(alpha)-Acetylation is one of the most common protein modifications in eukaryotes but a rare event in prokaryotes. Some endogenously N(alpha)-acetylated proteins in eukaryotes are frequently reported not to be acetylated or only very partially when expressed in recombinant Escherichia coli. Thymosin alpha1 (Talpha1), an N(alpha)-acetylated peptide of 28 amino acids, displays a powerful general immunostimulating activity. Here, we revealed that a fusion protein of thymosin alpha1 and L12 is partly N(alpha)-acetylated in E. coli. Through deletion of some N(alpha)-acetyltransferases by Red recombination, we found that, when rimJ is disrupted, the fusion protein is completely unacetylated. The relationship of rimJ and N(alpha)-acetylation of Talpha1 was further investigated by gene rescue and in vitro modification. Our results demonstrate that N(alpha)-acetylation of recombinant Talpha1-fused protein in E. coli is catalyzed by RimJ and that fully acetylated Talpha1 can be obtained by co-expressing with RimJ. This is the first description that an ectopic protein acetylation in bacterial expression systems is catalyzed by RimJ, a known prokaryotic N(alpha)-acetyltransferase.
N（α）-乙酰化是真核生物中最常见的蛋白质修饰之一，但在原核生物中则较为罕见。一些真核生物中内源性N（α）-乙酰化的蛋白质在重组大肠杆菌中表达时，经常被报道未被乙酰化或仅部分乙酰化。胸腺素α1（Tα1）是一种由28个氨基酸组成的N（α）-乙酰化肽，具有强大的一般免疫刺激活性。在此，我们发现胸腺素α1与L12的融合蛋白在大肠杆菌中部分N（α）-乙酰化。通过Red重组缺失一些N（α）-乙酰转移酶，我们发现，当rimJ被破坏时，融合蛋白完全未被乙酰化。通过基因拯救和体外修饰进一步研究了rimJ与Tα1的N（α）-乙酰化之间的关系。我们的结果表明，大肠杆菌中重组Tα1融合蛋白的N（α）-乙酰化由RimJ催化，并且通过与RimJ共表达可以获得完全乙酰化的Tα1。这是首次描述细菌表达系统中的异位蛋白质乙酰化由已知的原核N（α）-乙酰转移酶RimJ催化。
