固相合成蛋白所需生物大分子Linker-Met和fma-tRNA的构建(英文)

Construction of Linker-Met and Fma-t RNA for Immobilized Protein Synthesis

无细胞表达蛋白质体系近年来恢复活力,以满足日益增长的蛋白合成需求.与传统的细胞重组表达蛋白的方法相比,无细胞体系具有许多优点,包括更快的速度、更高的通量和更大的灵活性.此外,它提供了独特的可能性,如合理的DNA模板设计,毒素蛋白的表达,多个多肽链的联合表达,高效的定位标记等等.在本文中,建立了一个linker-tRNA的方法利用无细胞体系固相合成蛋白质.首先,需要将第1个氨基酸连接上起始tRNA通过linker连接到固相表面.为了提高固相合成蛋白质的效率,利用含有琥珀终止子(CUA)为反密码子的linker-tRNA与含互补琥珀子(UAG)为起始密码子的DNA模板而不是正常的起始密码子(密码子AUG).已经成功地获得了纯化的fma-tRNA(-CA)和linker-Met了.此方法将为蛋白质芯片的开发提供新途径.

Recent technical advances have revitalized cell-free expression systems to meet the increasing demands for protein synthesis.Cell-free systems offer several advantages over traditional cell-based expression methods,including greater speed,throughput and flexibility.In addition,it offers unique possibilities such as rational DNA template design,expression of toxic proteins,co-expression of multiple polypeptides,efficient site-specific labeling for NMR and X-ray studies,and rapid production of engineered protein variants.In this paper,we intend to construct a linkertRNA to develop a solid phase protein translation method which allows for the synthesis of proteins on an immobile phase using cell free systems.To achieve this,we need to link the first amino acid,which is coupled to an initiator tRNA,to the surface of a solid support through a linker.To improve the efficiency of protein synthesis on solid phase,we utilize an amber （CUA） initiator suppressor tRNA chemically aminoacylated with a linker modified amino acid and a DNA template containing a complementary amber （UAG） codon instead of the normal initiation （AUG） codon.We have successfully acquired the purified fma-tRNA（-CA） and the linker-Met.This method will provide a new way for the development of protein arrays.