Yates Elizabeth A, Estrella Luis A, So Christopher R
Department of Chemistry, US Naval Academy, Annapolis, MD, USA.
Formerly Chemistry Division, US Naval Research Laboratory, Washington, DC, USA.
Methods Mol Biol. 2022;2538:131-144. doi: 10.1007/978-1-0716-2529-3_10.
Escherichia coli remains one of the most widely used workhorse microorganisms for the expression of heterologous proteins. The large number of cloning vectors and mutant host strains available for E. coli yields an impressively wide array of folded globular proteins in the laboratory. However, applying modern functional screening approaches to interrogate insoluble protein aggregates such as amyloids requires the use of nonstandard expression pathways. In this chapter, we detail the use of the curli export pathway in E. coli to express a library of gene fragments and variants of a functional amyloid protein to screen sequence traits responsible for aggregation and the formation of nanoscale materials.
大肠杆菌仍然是用于表达异源蛋白的最广泛使用的主力微生物之一。可用于大肠杆菌的大量克隆载体和突变宿主菌株在实验室中产生了令人印象深刻的各种折叠球状蛋白。然而，应用现代功能筛选方法来研究不溶性蛋白质聚集体（如淀粉样蛋白）需要使用非标准表达途径。在本章中，我们详细介绍了利用大肠杆菌中的卷曲纤维输出途径来表达功能性淀粉样蛋白的基因片段文库和变体，以筛选负责聚集和纳米级材料形成的序列特征。
