Cell‐free complements in vivo expression of the E. coli membrane proteome

Abstract

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cellfree expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.Keywords: cell-free protein expression; integral membrane proteins; structural genomics; high-throughput protein expression Integral membrane proteins (MPs), despite their biological importance, currently account for <1% of all known high resolution protein structures. MPs are notoriously difficult to work with, and expression, detergent solubilization, purification, and crystallization all present unique challenges over their soluble counterparts (White 2004). MPs generally express at much lower levels than soluble proteins and, when in vivo overexpression is successful, the protein can be cytotoxic or incorporated into insoluble inclusion bodies. Following successful MP expression, a suitable detergent condition must also be found that simultaneously extracts the protein from the membrane while retaining the native fold and function. This protein-detergent complex (PDC) is often heterogeneous, creating numerous problems in purification and crystallization. Optimizing purification, assaying protein function, and crystallization all require milligram quantities of protein, and MP expression is therefore a limiting step in macromolecular structure determination (Dobrovetsky et al. 2005;Eshaghi et al. 2005;Korepanova et al. 2005;Columbus et al. 2006;Surade et al. 2006). One recognized alternative is cell-free (CF) expression (Klammt et al. 2004). 4 These authors contributed equally to this work.

Cotranslational cis -phosphorylation of the COOH-terminal tail is a key priming step in the maturation of cAMP-dependent protein kinase

“…His 6 PKAs were overexpressed in a continuous exchange cell-free (CF) system as previously described (25) with further optimization. In general, CF extracts were prepared from the E. coli strain A19 (25), and T7-RNA polymerase was expressed using the pT7-911Q plasmid (48) and purified as described (49). CF expression was conducted in dialysis mode using Mini Slide-A-Lyzers with 20 kDa MWCO (Thermo Scientific) with 200 μL reaction mixture and 1,000 μL feeding mixture.…”

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

“…However, these assays are difficult to develop for many membrane proteins (22,23) and particularly for olfactory receptors. A more feasible approach is to study the protein homogeneity by using size-exclusion chromatography (SEC).…”

High yield expression of proteins in &lt;i&gt;E. coli&lt;/i&gt; for NMR studies

“…those proteins where purification is not feasible like-membrane proteins which pose obstacles to work with [70], viral proteins [69], incorporation of non-natural amino acids [70,71], in studying protein-interactions and also in high throughput proteomics [69,72]. In addition, cell free method provides a means by which target proteins can be decisively screened for NMR studies by means of multiple screening systems [73].…”