TLR序列在SRP54蛋白与SRPRNA和信号肽结合中的作用

SRP54蛋白是信号识别颗粒(signal recognition particle)的一个关键组分.对人SRP54蛋白328～330位的TLR3个氨基酸进行人工诱变,在大肠杆菌BL21(DE3)pLysS中表达了A3突变体,并对A3突变体进行纯化和Superdex75凝胶过滤分析.观察到A3突变体丧失了与SRPRNA结合的能力,其自身也不能形成二聚体.结果证明,TLR这3个氨基酸残基与二聚体结构的形成有关,TLR是SRP54蛋白结合SRPRNA和新生蛋白质信号肽所必需的关键性氨基酸序列.

Role of TLR Sequence in Binding of SRP54 with SRP RNA and Signal Peptide

SRP54 protein is a crucial component of signal recognition particle (SRP). In this study, TLR, the three amino acid residues at positions 328—330 of human protein SRP54, were replaced with three alanine (A3 mutant) by using site-directed mutagenesis. A3 mutant was then transfected to express in E.coli BL21(DE3)pLysS, and the expressed A3 protein was purified by ion exchange chromatography and analysed further by Superdex 75 gel filtration,sequentially. The results showed that A3 mutant lost the ability to bind to SRP RNA and can not form dimeric structure by itself at 4℃. Our results demonstrated that TLR sequence at positions 328—330 was required for forming the dimeric structure of protein SRP54 at 4℃ and TLR was the essential amino acid sequence of human protein SRP54 which was also necessary for SRP54 binding to SRP RNA and signal peptide of nascent secretory protein.