Wadsworth Center, New York State Department of Health, Albany, New York.
Signature Science, LLC, Austin, Texas.
Curr Protoc. 2021 Mar;1(3):e82. doi: 10.1002/cpz1.82.
Inteins (intervening proteins) are translated within host proteins and removed through protein splicing. Conditional protein splicing (CPS), where the rate and accuracy of splicing are highly dependent on environmental cues, has emerged as a novel form of post-translational regulation. While CPS has been demonstrated for several inteins in vitro, a comprehensive understanding of inteins requires tools to quantitatively monitor their activity within the cellular context. Here, we describe a method for construction of a splicing-dependent system that can be used to quantitatively assay for conditions that modulate protein splicing. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Construction of an intein-containing KanR2 library using Gibson assembly Basic Protocol 2: Phenotype determination using quantitative spot titers Support Protocol 1: Preparation of LB agar plates for spot titers Support Protocol 2: Preparation and transformation of competent M. smegmatis cells.
内肽 ( intervening proteins) 在宿主蛋白内被翻译，并通过蛋白剪接去除。条件性蛋白剪接 (CPS) 是一种新型的翻译后调控方式，其剪接的速率和准确性高度依赖于环境线索。尽管已经在体外对几种内肽进行了 CPS 实验，但要全面了解内肽，还需要工具来定量监测它们在细胞环境中的活性。本文描述了一种构建依赖剪接的系统的方法，可用于定量测定调节蛋白剪接的条件。© 2021 威立出版公司。 基本方案 1：使用 Gibson 组装构建含内肽的 KanR2 文库 基本方案 2：使用定量点滴定法进行表型测定 支持方案 1：用于点滴定的 LB 琼脂平板的制备 支持方案 2：制备和转化感受态 M. smegmatis 细胞
