Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA.
The Nebraska Center for Integrated Biomolecular Communication (NCIBC), University of Nebraska-Lincoln, Lincoln, NE, USA.
Methods Mol Biol. 2024;2760:209-217. doi: 10.1007/978-1-0716-3658-9_13.
Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.
新兴微生物 Pseudomonas putida KT2440 可用于利用可再生原料合成生物基化学品和进行生物修复。然而，该生物体中生物合成酶和蛋白质复合物的分析、工程和调控方法仍欠发达。通过遗传密码扩展，将非天然氨基酸（ncAAs）掺入蛋白质中，可以进一步控制菌株的生物过程，从而推动这些方法的发展。在这里，我们将逐步介绍如何通过两种常用的正交古细菌 tRNA 合成酶和 tRNA 对，在 UAG 终止密码子的响应下，将两个非天然氨基酸掺入到任何感兴趣的蛋白质（POI）中。以超折叠绿色荧光蛋白（sfGFP）为例，该方法为未来研究和增强 KT2440 的生物学功能奠定了坚实的基础。
