Abstract
Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727–730
Li M, Hays FA, Roe-Zurz Z, Vuong L, Kelly L, Ho CM, Robbins RM, Pieper U, O'Connell JD 3rd, Miercke LJ, Giacomini KM, Sali A, Stroud RM (2009) Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening. J Mol Biol 385:820–830
Grisshammer R, Tate CG (1995) Overexpression of integral membrane proteins for structural studies. Q Rev Biophys 28:315–422
Magnani F, Shibata Y, Serrano-Vega MJ, Tate CG (2008) Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor. Proc Natl Acad Sci U S A 105:10744–10749
Elena C, Ravasi P, Castelli ME, Peiru S, Menzella HG (2014) Expression of codon optimized genes in microbial systems: current industrial applications and perspectives. Front Microbiol 5:21
Rosano GL, Ceccarelli EA (2009) Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microb Cell Fact 8:41
Vogl T, Thallinger GG, Zellnig G, Drew D, Cregg JM, Glieder A, Freigassner M (2014) Towards improved membrane protein production in Pichia pastoris: general and specific transcriptional response to membrane protein overexpression. N Biotechnol 31(6):538–552
Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673–681
Gourdon P, Liu XY, Skjorringe T, Morth JP, Moller LB, Pedersen BP, Nissen P (2011) Crystal structure of a copper-transporting PIB-type ATPase. Nature 475:59–64
Andersson M, Mattle D, Sitsel O, Klymchuk T, Nielsen AM, Moller LB, White SH, Nissen P, Gourdon P (2014) Copper-transporting P-type ATPases use a unique ion-release pathway. Nat Struct Mol Biol 21:43–48
Acknowledgements
This work was supported by the Graduate School of Science and Technology at Aarhus University and by grants to P.G. from the Swedish Research Council and the The Lundbeck Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Liu, X., Sitsel, O., Wang, K., Gourdon, P. (2016). Overproduction of PIB-Type ATPases. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3179-8_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
eBook Packages: Springer Protocols