Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.
Rationally evolving tRNAPyl for efficient incorporation of noncanonical amino acids.
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DOI：
10.1093/nar/gkv800
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Söll D
中科院分区：
文献类型：
--
作者：
Fan C;Xiong H;Reynolds NM;Söll D
将非经典氨基酸（ncAAs）基因编码到蛋白质中是研究蛋白质功能的一种有效方法。吡咯赖氨酰-tRNA合成酶（PylRS）是一种广泛使用的多特异性氨酰-tRNA合成酶，迄今为止，它已促进了大量不同ncAA掺入蛋白质中。为了使这一过程更有效，我们合理地进化了tRNAPyl，以创建具有六个核苷酸变化的tRNAPyl-opt。将这种改进的tRNA作为野生型PylRS以及三种表征的PylRS变体（N-乙酰基赖氨酰-tRNA合成酶[AcKRS]、3-碘-苯丙氨酰-tRNA合成酶[IFRS]、广泛特异性PylRS变体[PylRS-AA]）的底物进行测试，以在超级折叠绿色荧光蛋白（sfGFP）中的UAG密码子处掺入ncAA。tRNAPyl-opt促进AcK同时掺入sfGFP的两个位置的5倍增加。此外，AcK掺入到两个目标蛋白（大肠杆菌苹果酸脱氢酶和人组蛋白H3）引起高产量的多个赖氨酸残基的均匀乙酰化。使用tRNAPyl-opt与PylRS和各种PylRS变体促进了六种其他ncAA有效掺入sfGFP。动力学分析显示，tRNAPyl-opt中的突变对PylRS酶的催化效率和底物结合没有显著影响。因此，tRNAPyl-opt应该是野生型tRNAPyl的极好替代物，用于将来通过PylRS酶掺入ncAA。
Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNAPyl to create tRNAPyl-opt with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (Nϵ-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNAPyl-opt facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNAPyl-opt with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNAPyl-opt had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNAPyl-opt should be an excellent replacement of wild-type tRNAPyl for future ncAA incorporation by PylRS enzymes.