Ma C, Kudlicki W, Odom O W, Kramer G, Hardesty B
Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Biochemistry. 1993 Aug 10;32(31):7939-45. doi: 10.1021/bi00082a015.
The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli. DNA sequences similar to the natural tRNA(Ala/UGC) gene from E. coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase. The UGC alanine anticodon was changed to CUA corresponding to the UAG stop codon, CCU corresponding to the rarely used AGG arginine codon, and two four-nucleotide anticodons used to suppress stop codons. Bacterial dihydrofolate reductase was the test protein. Its cloned coding sequence was mutagenized at the GUG codon for valine-75 to correspond to the anticodons of the tRNA constructs, and then the plasmids were used to direct the synthesis of dihydrofolate reductase in the coupled transcription/translation system containing the corresponding synthetic tRNA. The results indicate that all four synthetic tRNAs were functionally active in the synthesis of full-length, enzymatically active dihydrofolate reductase protein.
在由大肠杆菌制备的偶联转录/翻译系统中，测试了合成tRNA用于体外蛋白质工程的情况。体外合成了与大肠杆菌天然tRNA（Ala/UGC）基因相似但反密码子不同的DNA序列，将其克隆到DNA质粒中，然后用T7 RNA聚合酶进行体外转录。UGC丙氨酸反密码子被改为对应UAG终止密码子的CUA、对应很少使用的AGG精氨酸密码子的CCU，以及用于抑制终止密码子的两个四核苷酸反密码子。细菌二氢叶酸还原酶是测试蛋白。其克隆的编码序列在缬氨酸-75的GUG密码子处诱变，以对应tRNA构建体的反密码子，然后将质粒用于在含有相应合成tRNA的偶联转录/翻译系统中指导二氢叶酸还原酶的合成。结果表明，所有四种合成tRNA在全长、具有酶活性的二氢叶酸还原酶蛋白的合成中均具有功能活性。
