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The Funnel Approach to the Precrystallization Production of Membrane Proteins

Lewinson, Oded and Lee, Allen T. and Rees, Douglas C. (2008) The Funnel Approach to the Precrystallization Production of Membrane Proteins. Journal of Molecular Biology, 377 (1). pp. 62-73. ISSN 0022-2836. PMCID PMC2362151. doi:10.1016/j.jmb.2007.12.059.

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Challenges in the production of integral membrane proteins for structural studies include low expression levels, incorrect membrane insertion, aggregation and instability. In this report, we describe a “funnel approach” to overcoming these difficulties and demonstrate its efficacy in a case study of 36 prokaryotic P-type transporters. A diverse ensemble of modified constructs is generated and tested for expression in Escherichia coli, membrane localization, detergent extraction, and homogeneity. High-throughput methodologies are implemented throughout the process to facilitate identification of promising targets. We find that the choice of promoter, the choice of source organism providing the cloned gene, and, most importantly, the position of the affinity tag have a great effect on successful production. The latter had pronounced effects at all tested levels, from expression levels observed in whole cells to the extent of membrane insertion, and even on protein function. Following the initial streamlined screening, we were able to fine-tune and produce 9 of the 36 targets as materials suitable for crystallization or other structural studies.

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Rees, Douglas C.0000-0003-4073-1185
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Additional Information:© 2007 Elsevier Ltd. All rights reserved. Received 23 September 2007; received in revised form 20 December 2007; accepted 21 December 2007 The authors would like to thank members of the Rees group for their help in the preparation of this manuscript. We thank Dietrich H. Nies for the generous gift of the plasmid DNA of R. metallidurans genes; Chris Rensing for strains W3110 and GG48; David B. Wilson for the plasmid DNA of Lactobacillus plantarum mntA; B. Sarkar for the plasmid DNA of Cu(6)-ZntA and Cu(1–6)–zntA; Bharati Mitra for E. coli zntA; and Gianluca Quintini for H. pylori CadA. This work was supported, in part, by grants from the Fulbright Foundation and the Jane Coffin Childs Memorial Fund for Medical Research to O.L.
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Fulbright FoundationUNSPECIFIED
Jane Coffin Childs Memorial Fund for Medical ResearchUNSPECIFIED
Subject Keywords:membrane protein; crystallization; P-type ATPase
Issue or Number:1
PubMed Central ID:PMC2362151
Record Number:CaltechAUTHORS:20141212-143814641
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52777
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Deposited On:22 Dec 2014 21:16
Last Modified:10 Nov 2021 19:43

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