重组人蛋白的筛选与生产：连接酶独立克隆。

Screening and Production of Recombinant Human Proteins: Ligation-Independent Cloning.

机构信息

Diamond Light Source Ltd., Didcot, Oxfordshire, UK.

Astex Pharmaceuticals, Cambridge, UK.

出版信息

Methods Mol Biol. 2021;2199:23-43. doi: 10.1007/978-1-0716-0892-0_3.

DOI:10.1007/978-1-0716-0892-0_3

PMID:33125643

Abstract

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).

摘要

结构基因组学研究小组已经认识到，需要生成每个目标的多个截断版本，以提高生成表达良好、可溶性和稳定性蛋白质的成功率，并且该蛋白质能够结晶并衍射到足以进行结构确定的分辨率。在结构基因组学联合会，我们选择了连接酶独立克隆（LIC）方法，该方法提供了我们所期望的高通量，以在平行过程中生产和筛选许多蛋白质。在这里，我们描述了用于生成 96 孔格式构建体的 LIC 方案，并提供了适合在大肠杆菌和杆状病毒表达载体系统（BEVS）中表达蛋白质的载体选择。