Kim Sanggil, Lee Hyun Soo
Department of Chemistry, Sogang University.
Department of Chemistry, Sogang University;
J Vis Exp. 2018 Aug 24(138):58383. doi: 10.3791/58383.
L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical properties, the amino acid has multiple uses in biochemical applications. This report describes a protocol for the genetic incorporation of biosynthesized DOPA and its application to protein conjugation. DOPA is biosynthesized by a tyrosine phenol-lyase (TPL) from catechol, pyruvate, and ammonia, and the amino acid is directly incorporated into proteins by the genetic incorporation method using an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase pair. This direct incorporation system efficiently incorporates DOPA with little incorporation of other natural amino acids and with better protein yield than the previous genetic incorporation system for DOPA. Protein conjugation with DOPA-containing proteins is efficient and site-specific and shows its usefulness for various applications. This protocol provides protein scientists with detailed procedures for the efficient biosynthesis of mutant proteins containing DOPA at desired sites and their conjugation for industrial and pharmaceutical applications.
L-二羟基苯丙氨酸（DOPA）是一种存在于动植物儿茶酚胺生物合成过程中的氨基酸。由于其特殊的生化特性，该氨基酸在生化应用中有多种用途。本报告描述了一种生物合成DOPA的基因掺入方法及其在蛋白质偶联中的应用。DOPA由酪氨酸酚裂解酶（TPL）从儿茶酚、丙酮酸和氨生物合成，并且通过使用进化的氨酰-tRNA和氨酰-tRNA合成酶对的基因掺入方法将该氨基酸直接掺入蛋白质中。这种直接掺入系统能够高效地掺入DOPA，几乎不掺入其他天然氨基酸，并且与先前用于DOPA的基因掺入系统相比，蛋白质产量更高。含DOPA的蛋白质的蛋白质偶联是高效且位点特异性的，并显示出其在各种应用中的实用性。该方案为蛋白质科学家提供了详细的程序，用于在所需位点高效生物合成含DOPA的突变蛋白及其在工业和制药应用中的偶联。
