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. 2013 Nov 5:74:29.6.1-29.6.34.
doi: 10.1002/0471140864.ps2906s74.

High-throughput cloning and expression of integral membrane proteins in Escherichia coli

Renato Bruni  1 , Brian Kloss  1
Affiliations

High-throughput cloning and expression of integral membrane proteins in Escherichia coli

Renato Bruni et al. Curr Protoc Protein Sci. .

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 the laboratory. Basic Protocol 1 describes 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 do express on the miniscale, Basic Protocols 3 and 4 outline the methods employed for the expression and purification of targets on a midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable.

Keywords: Escherichia coli; cloning/expression; high-throughput; ligation-independent cloning (LIC); membrane protein; membrane protein purification; recombinant protein expression.

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Figures

Figure 1
Figure 1
Flowchart outlining the methods described in this unit. Part 1: Amplification and cloning of targets. Part 2: Expression and miniscale purification of target proteins. Part 3: Scale-up and detergent screening.
Figure 2
Figure 2
Schematic outline of the four expression vectors used by NYCOMPS. Only relevant features are shown. lacI: lac repressor. RBS: ribosomal binding site. FLAG: DYKDDDDK epitope. 10×His: decahistidine tag. TEV: tobacco etch virus protease recognition site (ENLYFQS). 5′ and 3′ LIC: overhang sites for ligation-independent cloning. ccdB: CcdB toxin gene for plasmid maintenance. Restriction enzymes for linearizing the vectors for LIC treatment are also shown. Features are not to scale.
Figure 3
Figure 3
Coomassie-stained gel of proteins expressed and purified at the small scale. 24 unique targets were grown in 1 ml media in a 96 well deep-well block and purified by Ni2+ affinity chromatography. A portion of each purified protein was separated by SDS-PAGE and visualized by staining with Coomassie blue. Sizes of molecular weight standards are indicated along the left hand side of the gel. Contaminating proteins are indicated by arrowheads. Expressed proteins are indicated by asterisks.
Figure 4
Figure 4
Assembly of the GNF Fermenter. The photo in Panel A shows the 96-pronged replicating tool from Enzyscreen. The tips of the tool are washed with the brush under deionized water and sterilized by heating to 300°C on a hot plate for 5 minutes. 8-10 ml 2×TY in each of the 96 tubes of the GNF Fermenter are inoculated from glycerol stocks and the entire rack is loaded into an Innova 44 shaker and grown to saturation overnight at 37°C as shown in Panel B. The following morning, fresh media is added to each tube with the aid of a peristaltic pump as shown in Panel C. Panel D shows the gas manifold. The manifold sits on top of the fermenter tubes, with each of the 96 cannulae delivering air and O2 directly into each culture. This provides exceptional aeration and mixing. Panel E shows the completely assembled fermenter inside a fume hood. The rack of tubes is lowered into a circulating water bath for temperature control. The gas mixing chamber and controller are to the right of the fermenter. Panel F shows the media before (left) and after (right) the 5.5 hour growth and induction protocol. Optical densities, measured at 600 nm, of greater than 20, are routinely obtained.
Figure 5
Figure 5
Coomassie-stained gel of proteins expressed and purified at the mid-scale. 24 unique targets were grown in the GNF Fermenter and purified from 50 ml of fermenter culture. Approximately 10% of each purified protein was loaded on to a gel and visualized by staining with Coomassie blue. Molecular weight standards are indicated along the left side of the gel. Half of each purified protein was immediately analyzed by size exclusion chromatography in DDM. Gel filtration traces of two of the 24 purified proteins are shown. Both proteins are monodisperse and are excellent candidates for further analysis.
Figure 6
Figure 6
Detergent stability assay results. Three different proteins were purified in DDM, divided into four portions and mixed with a large excess of LDAO, C8E4, or βOG. The addition of an excess quantity of the same detergent used for purification, DDM, serves as a control. Panel A shows a protein that is well behaved in DDM, but no others. The protein in Panel B tolerates LDAO and βOG, although there is likely some protein loss, as seen by the lower protein peak height as compared to that seen in DDM. The same protein is completely lost when combined with C8E4. It most likely precipitated during incubation at room temperature and was removed by filtration prior to size exclusion chromatography. Panel C is well behaved in all four detergents and is an excellent candidate for scale-up and crystallographic studies.
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