Functional in vitro and in vivo analysis of biosynthetic genes by heterologous expression in E. coli

• Published in: STAR protocols Vol. 4; no. 3; p. 102531
• Main Authors: Kramer, Jil-Christine, Böhringer, Nils, Mettal, Ute, Schäberle, Till F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-09-2023; Elsevier
• Subjects: Biological Products - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Genetics; Metabolomics; Multigene Family - genetics; Protein Expression and Purification; Protocol; Metabolomics; Genetics; Protein Expression and Purification
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102531

Biosynthetic gene clusters of natural products often harbor genes of unknown function, which are difficult to characterize. Here, we present a protocol for the functional analysis in vitro and in vivo of these biosynthetic genes by heterologous expression in E. coli. We describe steps for the expression of genes of interest in an established E. coli strain optimized to heterologously express natural products. We then detail the expression of a His-tagged gene to deduce the specific function of the protein.
For complete details on the use and execution of this protocol, please refer to Böhringer et al.1
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•Explanation of primer design to clone a gene of interest by Gibson assembly•Heterologous expression of cloned genes of interest•Purification of new proteins with unknown function by His-tag affinity chromatography•In vitro assay to test the activity of the expressed protein in vitro
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Biosynthetic gene clusters of natural products often harbor genes of unknown function, which are difficult to characterize. Here, we present a protocol for the functional analysis in vitro and in vivo of these biosynthetic genes by heterologous expression in E. coli. We describe steps for the expression of genes of interest in an established E. coli strain optimized to heterologously express natural products. We then detail the expression of a His-tagged gene to deduce the specific function of the protein.