Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

Boggavarapu, Venkata Ravi Naga Rajendr; Hirschi, Stephan; Harder, Daniel; Meury, Marcel; Ucurum Fotiadis, Zöhre; Bergeron, Marc; Fotiadis, Dimitrios José (2016). Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies. In: Mus-Veteau, Isabelle (ed.) Heterologous Expression of Membrane Proteins. Methods and Protocols. Methods in Molecular Biology: Vol. 1432 (pp. 223-242). New York: Springer 10.1007/978-1-4939-3637-3_14

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This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.

Item Type:

Book Section (Book Chapter)

Division/Institute:

04 Faculty of Medicine > Other Institutions > NCCR TransCure
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Boggavarapu, Venkata Ravi Naga Rajendr; Hirschi, Stephan; Harder, Daniel; Meury, Marcel; Ucurum Fotiadis, Zöhre; Bergeron, Marc and Fotiadis, Dimitrios José

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

1064-3745

ISBN:

978-1-4939-3637-3

Series:

Methods in Molecular Biology

Publisher:

Springer

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

19 Apr 2017 09:58

Last Modified:

01 May 2017 15:34

Publisher DOI:

10.1007/978-1-4939-3637-3_14

PubMed ID:

27485339

Uncontrolled Keywords:

2D crystallization; Human; Mammalian; Membrane protein; Protein structure; Single particle analysis

BORIS DOI:

10.7892/boris.94001

URI:

https://boris.unibe.ch/id/eprint/94001

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