Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system
Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system
复制标题
DOI：
10.1263/jbb.102.511
复制
发表时间：
2006-12-01
影响因子：
2.8
通讯作者：
Sisido, Masahiko
中科院分区：
文献类型：
--
作者：
Taki, Masumi;Tokuda, Yasunori;Sisido, Masahiko
将草地贪夜蛾21（Spodoptera frugiperda 21，Sf 21）昆虫无细胞蛋白合成系统扩展到包括非天然氨基酸。探讨了在昆虫系统中作为非天然氨基酸载体的正交tRNA。还选择了用于分配非天然氨基酸位置的四碱基密码子。制备含有不同四碱基密码子的突变的链霉亲和素mRNA，并在具有相应四碱基反密码子的各种tRNA存在下将其添加到昆虫无细胞系统中。用各种类型的非天然氨基酸对tRNA进行化学氨酰化，以检查其掺入效率。以对硝基苯丙氨酸为非天然氨基酸，链霉亲和素为靶蛋白，优化tRNA序列和四碱基密码子类型，使非天然突变体产量最大化，不含非天然氨基酸的全长蛋白产量最小化。在从各种非标准结构的tRNA中提取的tRNA序列中，来自乙酸甲烷八叠球菌tRNA（Pyl）的tRNA是最有效和正交的tRNA。在CGGN型四碱基密码子中，CGGA和CGGG对非天然氨基酸的定位效率最高。对硝基苯丙氨酸和2-萘基丙氨酸被有效地纳入大肠杆菌和兔网织红细胞无细胞系统的情况下。然而，对于其他非天然氨基酸，观察到的掺入效率要低得多，这表明昆虫系统对氨基酸结构多样性的耐受性低于E. coli无细胞体系。
Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically amino-acylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.