Báo cáo khoa học: Evaluation of detergents for the soluble expression of a-helical and b-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system

Evaluation of Detergents for the Soluble Expression of α-Helical and β-Barrel-Type Integral Membrane Proteins by a Preparative Scale Individual Cell-Free Expression System

Authors:

Christian Klammt, Daniel Schwarz, Klaus Fendler, Winfried Haase, Volker Dötsch, and Frank Bernhard

Field:

Biophysical Chemistry, Structural Biology, Molecular Biology

Document Content:

This document details a study investigating the effectiveness of various detergents in facilitating the soluble expression of integral membrane proteins (IMPs) using a cell-free (CF) expression system. The research explores how different detergents impact the production, solubility, and activity of three structurally distinct IMPs: the bacterial α-helical multidrug transporter (EmrE), the β-barrel nucleoside transporter (Tsx), and the porcine vasopressin receptor (V2R), a eukaryotic G-protein coupled receptor. The study evaluates detergents for their ability to either resolubilize IMP precipitates or enable direct soluble expression. Findings indicate that specific classes of detergents, particularly long-chain polyoxyethylene-alkyl-ethers, are generally effective for the soluble expression of diverse IMPs. The research also highlights that the choice and concentration of detergent significantly influence the yield and functional folding of the expressed proteins. This work extends the application of CF expression to β-barrel type proteins, offering a method for efficient production of soluble and functional IMPs for structural and functional analyses.

Detailed Table of Contents:

• Introduction
• Keywords
• Cell-free expression of EmrE, Tsx and V2R as precipitates
• Cell-free expression of EmrE, Tsx and V2R in the presence of detergents
• Results
• Secondary structure analysis of the V2R and Tsx proteins and reconstitution into proteoliposomes
• Functional folding of the CF expressed nucleoside transporter, Tsx, depends on its mode of expression
• Discussion
• Experimental procedures
• DNA techniques
• Black lipid transport assay
• Cell-free expression
• CD spectroscopy
• Preparation and analysis of proteoliposomes
• Analysis and purification of proteins
• Acknowledgements
• References