Selection of Escherichia coli expression systems.
This unit lists the most useful expression strains of E. coli for fermentation processes. Standard procedures are provided for several expression systems, namely, temperature induction via the p(L) promoter and chemical induction via the trp promoter, lac or tac promoters, and the T7 promoter. These protocols require that the gene encoding the protein of interest has been identified and cloned into an appropriate expression vector using standard molecular biology techniques. Transformation of a suitable host strain (e.g., by electroporation) is also described and is a prerequisite. Protocols for the analysis of plasmid stability and subsequent storage are provided. Support protocols describe how to prepare samples for electrophoresis, how to analyze the solubility of the expressed proteins, and how to make samples of periplasmic extracts and extracellular media (using TCA precipitation). Many of the support protocols are small-scale analysis procedures that are used to guide subsequent purification strategies and determine the suitability of the expression system for further development and scale-up.
Bernard
A
A
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Payton
M
M
D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*
D011506:Proteins; D011994:Recombinant Proteins