Optimization of Membrane Protein Production Using Titratable Strains of E. coli

Abstract

The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.

Keywords: Green fluorescent protein (GFP); Membrane protein; RiboTite; Riboswitch; T7 RNA polymerase; T7 lysozyme; Titratable.