Purification of Membrane Proteins by Affinity Chromatography with On-Column Protease Cleavage.

Hirschi, Stephan; Fotiadis, Dimitrios (2020). Purification of Membrane Proteins by Affinity Chromatography with On-Column Protease Cleavage. In: Perez, Camilo; Maier, Timm (eds.) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology: Vol. 2127 (pp. 139-150). Humana Press/Springer 10.1007/978-1-0716-0373-4_10

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A protocol is described for the isolation of recombinant polyhistidine-tagged membrane proteins from overexpressing Escherichia coli cells. The gene encoding a target membrane protein is cloned into an expression plasmid and then introduced into E. coli cells for overexpression. Membranes from bacterial cells are isolated and the tagged target membrane protein is solubilized in detergent and subsequently bound to an affinity matrix. Tagged proteins are commonly eluted by an excess of a solute that competes for the binding to the matrix. Alternatively, amino acid sequence-specific proteases can be used to cleave off the affinity purification tag directly on the purification column (i.e., on-column cleavage). This selectively releases the target protein and allows subsequent elution. Importantly, this step represents an additional purification step and can significantly increase the purity of the isolated protein.

Item Type:

Book Section (Book Chapter)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Hirschi, Stephan and Fotiadis, Dimitrios José

Subjects:

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

ISSN:

1940-6029

ISBN:

978-1-0716-0372-7

Series:

Methods in Molecular Biology

Publisher:

Humana Press/Springer

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

06 Jul 2020 08:05

Last Modified:

11 Aug 2020 03:11

Publisher DOI:

10.1007/978-1-0716-0373-4_10

PubMed ID:

32112320

Uncontrolled Keywords:

Affinity chromatography Membrane protein On-column cleavage Protease cleavage Protein affinity tag

BORIS DOI:

10.7892/boris.144781

URI:

https://boris.unibe.ch/id/eprint/144781

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