An <i>Escherichia coli</i>-Based Cell-Free System for Large-Scale Production of Functional Mammalian Membrane Proteins Suitable for X-Ray Crystallography

Abstract

A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction ‘mode A’ (proteins were expressed as precipitant) and ‘mode B’ (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of ‘mode B’ and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.

Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins

“…The D-CF mode appears to have certain advantages as it (i) avoids any initial precipitation of the synthesised MPs, (ii) provides an efficient universal solubilisation approach independent of specific translocation systems, and (iii) delivers MP samples that can be used directly for a variety of functional and structural assays (e.g. [1,2,11,25,39,40]). Detergents used for the D-CF mode have to be in particular very mild as they must not affect any enzymes involved in the transcription/translation machinery.…”

“…Proteolytic degradation could further contribute to sample heterogeneity. The protein integrity in suspected bands after SDS gel analysis should therefore always be verified by subsequent detection of N-or/and C-terminally attached immunotags [1][2][3][4][5]11,[24][25][26]28,39,40,43,56,[62][63][64]. MALDI-TOF mass spectrometry can be considered a complementary tool to determine the mass and the identity of the synthesised proteins [3,11,63].…”

“…Oligomerisation has been assessed for CF-produced MPs by pull-down studies of differently tagged rat ion transporters [24], by co-expression of EmrE species [2] or by cross-linking as reported for MscL [1,62] and [2] EmrE. Sample staining techniques followed by electron microscopy analyses have been successfully applied to demonstrate oligomer formation of the CF-expressed bacterial outer membrane secretin PulD [56], of different GPCRs [40,65], of the mechanosensitive channel MscL [62], as well as of the human MP CX32 [39] or the formation of nanodiscs [69].…”

“…A fast and preliminary evaluation of a-helical or b-sheet secondary structural elements can be done by circular dichroism (CD) spectroscopy [5,11,24,39,63,66]. This technique determines the overall content of folded protein parts and gives first indications of expression conditions providing suitable folding conditions.…”

“…Alternatively, ligand binding activities and specific complex formation may be investigated in proteoliposomes as well as in detergent micelles. CF expression has been used for a wide spectrum of MPs so far, spanning from toxic targets like the b-subunit of ATP synthase [74], b-barrel MPs like VDAC [39,75] and Tsx [5], to even large MPs with numerous transmembrane alpha helices [24,25,28,40,52,66]. The list of functionally characterised CF expressed MPs is thus continuously growing and already comprises a comprehensive selection of prokaryotic as well as eukaryotic targets of diverse kind of functions.…”