High‐Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

Abstract

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable.

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

“…16 1 Table S1. Cryo-EM data collection and modeling statistics WT-AftD, Related to Figure 2 1 2 This study (Bruni and Kloss, 2013) M. abscessus AftD-K39A in pNYCOMPS-N23…”

“…Indeed, this observation was corroborated by measurements of 24 the cell length and width, showing a statistically significant reduction in cell length of the cKO 1 strain in the presence (1.9 ± 0.6 μ m) versus the absence (2.5 ± 0.8 μ m) of tetracycline, while the 2 widths remained the same ( Figure 1H). 3 4 Genomic Expansion and Complementation of the M. abscessus aftD Gene 5 To study the AftD function in vitro and determine its structure, we adopted a structural genomics 6 approach (Bruni and Kloss, 2013;Love et al, 2010;Mancia and Love, 2010) to identify homologs 7 that yielded high expression levels in E. coli and were stable in detergents compatible with structure 8 determination (see Methods). High-throughput small-scale expression and purification screens of 9…”

“…previous protocol byBruni & Kloss (Bruni and Kloss, 2013). Only one construct, M. abscessus 11AftD in the pNYCOMPS-N23 plasmid, was successfully cloned and expressed in small and 12 medium scale.…”

Atomic-level analysis of membrane-protein structure

“…Efficient procedures have been developed for screening for the expression of membrane proteins in bacterial systems 9 , although their effectiveness for eukaryotic proteins has been limited. Various alternatives are in common use for the recombinant production of eukaryotic proteins, usually from synthetic genes, with a focus on baculovirus-infected insect cells and on cultures of mammalian cells, typically human embryonic kidney cells, HEK293.…”

Rapid automated detergent screening for the solubilization and purification of membrane proteins and complexes

“…So far, 491 unique integral membrane proteins have been solved by X‐ray crystallography, as mentioned in the Membrane Proteins of Known Structure Database (http://blanco.biomol.uci.edu/mpstruc/), whereas 2222 nonunique structures of integral membrane proteins (1928 alpha‐ and 294 beta‐folded) are listed in the Membrane Protein Data Bank (http://pdbtm.enzim.hu/). The number of membrane protein structures is expected to increase significantly in the coming years, particularly thanks to recently developed high‐throughput facilities .…”