Fluorescent In Situ Folding Control for Rapid Optimization of Cell-Free Membrane Protein Synthesis

Annika Müller-Lucks,Sinja Bock,Binghua Wu,Eric Beitz,Petri Kursula

doi:10.1371/journal.pone.0042186

Abstract

Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

Highlights

The cloning of the green fluorescent protein (GFP) gene [1] has initiated a multitude of applications
Constructs are generated of the test protein with GFP fused to its C-terminus and formation of the GFP fluorophore is monitored as a measure of the amount of correctly folded protein, see Drew et al for protocols [3]
Synthesis of PfAQP-GFP in an E. coli S30 extract-based cell-free system [7] using a panel of five typical non-ionic detergents for membrane protein solubilization [8] produced two bands with apparent molecular weights of 45 and 48 kDa as detected by an anti-GFP antiserum (Fig. 1A, left panel; Fig. S1)

Summary

Introduction

One is its use as a ‘‘folding indicator’’, which has been intended to evolve proteins by mutation for improved expression in Escherichia coli [2] To this end, constructs are generated of the test protein with GFP fused to its C-terminus and formation of the GFP fluorophore is monitored as a measure of the amount of correctly folded protein, see Drew et al for protocols [3]. Optimization of the reaction protocol, e.g. selection of a suitable detergent for protein solubilization, is limited by lengthy procedures of protein purification and structural or functional analysis to assay for correct protein folding. For this reason, cell-free synthesis conditions are usually optimized with the focus on protein yield, yet without knowledge of the folding status

Methods

Results

Conclusion