Comparative analysis and “expression space” coverage of the production of prokaryotic membrane proteins for structural genomics

Surade, Sachin; Klein, Markus; Stolt-Bergner, Peggy; Muenke, Cornelia; Roy, Ankita; Michel, Hartmut

doi:10.1110/ps.062312706

Cited by 57 publications

(44 citation statements)

References 39 publications

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“…It is also striking that the two systems can cumulatively express 75% of all targets. Lack of expression is the first bottleneck in structural studies of MPs, and obtaining 75% of the desired targets is in line with the best current published reports (Eshaghi et al 2005;Surade et al 2006). Finally, 30% (36 out of 120) of the targets could be expressed in either system, suggesting there is significant overlap in the abilities of the two systems to express targets.…”

Section: Cell-free Complements In Vivo Expressionsupporting

confidence: 80%

“…Overall, 63% of proteins expressed in CF, while only 44% of proteins expressed in vivo. Thus, we can express the majority of E. coli MPs selected for this study, and combined use of the two systems results in increased coverage of ''expression space'' (Surade et al 2006). Given the large number of successful expressers, we next sought to characterize the detergent solubility of these proteins and their potential for purification.…”

Section: Cell-free and In Vivo Protein Expression Resultsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

et al. 2007

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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.

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Section: Cell-free Complements In Vivo Expressionsupporting

confidence: 80%

Section: Cell-free and In Vivo Protein Expression Resultsmentioning

confidence: 99%

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

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“…21. Equal concentrations of membranes from each construct were analyzed by SDS-PAGE and Western blot using an ␣-His-HRP-conjugated antibody (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues

Malle

Zhou

Neuhold

et al. 2011

Journal of Biological Chemistry

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The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di-and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT so , identifies Glu 56 and Arg 305 as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.

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“…The authors further speculated that low yields of recombinant membrane proteins might be due to limited Sec translocon capacity. A second prokaryote, the Gram-positive bacterium Lactococcus lactis, has also been used to produce a wide range of eukaryotic and prokaryotic membrane proteins [16], enabling a comparison of L. lactis and E. coli [17]. Although a large fraction of proteins could be produced in both hosts, some could only be produced in one or the other.…”

Section: The Biosynthesis Of Recombinant Membrane Proteinsmentioning

confidence: 99%

Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

Ashe

Bill

2011

Biotechnology Journal

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Membrane proteins are targeted by over 50 % of marketed pharmaceuticals, and as most membrane proteins are not naturally abundant, they must be produced recombinantly for the structural biology that is a pre-requisite to structure-based drug design. Unfortunately, obtaining high yields of functional, recombinant membrane proteins remains a major bottleneck in contemporary bioscience. Trial-and-error optimisation studies have not (and cannot) reveal mechanistic details of the biology of recombinant protein production, highlighting the need for defined and rational optimisation strategies based upon experimental observation. To this end, we have published a transcriptome and subsequent genetic analysis that has identified genes implicated in high-yielding yeast cell factories. These results have highlighted a role for alterations to a cell's protein synthetic capacity in the production of high yielding cells: paradoxically, reduced protein synthesis favors higher yields of recombinant membrane protein. These results highlight a potential bottleneck at the protein folding or translocation stage of protein production.

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Comparative analysis and “expression space” coverage of the production of prokaryotic membrane proteins for structural genomics

Cited by 57 publications

References 39 publications

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

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

Random Mutagenesis of the Prokaryotic Peptide Transporter YdgR Identifies Potential Periplasmic Gating Residues

Mapping the yeast host cell response to recombinant membrane protein production: Relieving the biological bottlenecks

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