Membrane protein solubilization is achieved using wheat-germ cell-free translation by co-expression of the target membrane protein with a membrane scaffold protein derived from apolipoprotein in the presence of excess lipids. Two transcripts are added in varying ratios to optimize the co-translational formation of the soluble MSP/membrane protein complex. This complex can be purified using an affinity tag on either the IMP or the MSP. Soluble IMP/MSP complexes can be used for functional studies, characterization of membrane protein complexes, and the development of crystallography reagents such as synthetic antibody scaffolds.
Integral membrane proteins must be solubilized prior to purification, typically using detergents which can interfere with function and destabilize membrane protein structure. Here, we report the application of a detergent-free approach to membrane protein solubilization using wheat-germ cell-free expression. Nanodiscs, sometimes called nanolipoprotein particles, consist of a raft of lipid bilayer surrounded by two copies of an engineered amphiphilic helical section of apolipoprotein, called a membrane scaffold protein (MSP). The best characterized of MSPs derived from human apolipoprotein A1 and were developed by Drs. Stephen Sligar and colleagues (Bayburt and Sligar, 2010). The standard method of creating nanodiscs involves first purifying membrane protein in detergent, and combining precise ratios of the IMP, purified soluble MSP, and prepared lipids in a strong detergent such as sodium cholate. The detergents are removed using a detergent-removal resin, and the protein:lipid complex self assembles with the membrane protein embedded in the lipid bilayer, surrounded by the MSPs.
TMPC has adopted a simpler method of nanodisc self-assembly first described in Cappuccio, et al., 2008. In this approach, cell-free co-expression of IMPs and MSPs in the presence of lipids results in a solubilized membrane protein that can be purified with detergents and used for functional applications or antibody development. We have employed this approach using our wheat-germ cell-free expression platform, which uses uncoupled transcription and translation. With this system, we can adjust the ratio of each transcript added to translation to optimize the production of the soluble IMP/MSP complexes. We have also added a variety of tags on both the MSP and IMP genes to allow effective purification of native membrane proteins, and to increase the utility of this IMP preparation. A variety of different types of membrane proteins have been solubilized by this method (Figure 3A), including a complex of two different membrane proteins in a single nanodisc (Figure 3B). These preparations are routinely supplied to our collaborators for activity testing, and are also used for development of crystallization scaffold antibodies for use in structure determination of detergent-prepared IMPs.
Authors: Beebe ET, Makino S, Frederick RO, Fox BG
(A) Diagram of an assembled nanodisc. A multipass integral membrane protein (green) is embedded in a lipid bilayer raft (orange), surrounded by two copies of amphiphilic MSPs (blue). The hydrophilic sides of the MSP helices are toward the solvent, effectively solubilizing the membrane protein. Self-assembly occurs co-translationally. Solubilization and purification of IMPs using nanodiscs. Both panels (B) and (C) are tryptophan fluorescence images of SDS-PAGE gels. (B) Coexpression of human Sigma1 receptor (S1R), Trypanosoma sphingolipid synthase 3 (SLS3), and human stearoyl-CoA-desaturase 5 (SCD5) with scaffold proteins MSP1D1 (D1) or MSP1E3D1 (E3). E3 can form slightly larger nanodiscs to accommodate larger or multiple membrane proteins (Bayburt and Sligar, 2010). These MSPs do not contain affinity tags and are completely soluble. The IMPs are insoluble by themselves, but are solubilized by co-expression with MSPs. S, soluble fraction; P, pellet fraction. (C) IMAC purification of a complex between two membrane proteins after triple co-expression with MSP. Cox10 from either human (h10) or the Bacillus ortholog (b10) were co-expressed in combination with MSP and/or, in the case of Bacillus, with the interacting partner Cox15 (b15). Only one member of each co-expression contained a 6xHis tag (H6). Both D1 and E3 MSPs were tried. Note that b10 and b15 are similar sizes and in some combinations the untagged and H6-tagged proteins migrate at the same place on this gel.
Bayburt TH, Sligar SG. Membrane protein assembly into Nanodiscs. FEBS Lett 2010, 584(9):1721-7. PubMed ID:19836392
Cappuccio JA, Blanchette CD, Sulchek TA, Arroyo ES, Kralj JM, Hinz AK, Kuhn EA, Chromy BA, Segelke BW, Rothschild KJ, Fletcher JE, Katzen F, Peterson TC, Kudlicki WA, Bench G, Hoeprich PD, Coleman MA. Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles. Mol Cell Proteomics 2008, 7(11):2246-2253. PubMed ID:18603642 | PMC Link
Brian G. Fox (email@example.com)
Transmembrane Protein Center
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Last edited:Mon 10 Mar 2014 - 5 years, 1 month ago