Contribution to a book

PUBDB-2020-04434

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Transient expression of recombinant membrane-eGFP fusion proteins in HEK293 cells

Pieprzyk, J.Extern* ; Pazicky, S.CSSB* ; Loew, C. (Corresponding author)Extern*CSSB*

2018
Springer Science + Business Media LLC Berlin Heidelberg
ISBN: 978-1-4939-8730-6, 978-1-4939-8730-6 (electronic)

This record in other databases:      

Please use a persistent id in citations: doi:10.1007/978-1-4939-8730-6_2

Abstract: Membrane proteins play important roles in many biological processes and are a major drug target. However, only a limited number of structures of eukaryotic membrane proteins have been determined so far. Besides the challenges in crystallizing these proteins, one of the main bottlenecks in structure determination is their recombinant expression. The mammalian HEK293 cell line provides a natural environment for expression of eukaryotic membrane proteins but optimization of transfection and cultivation time is often necessary to yield amounts of protein suitable for structural studies. Here we describe a detailed protocol for expression and purification of membrane proteins from HEK293 cells with an example of the human peptide transporter, PepT2. In the first part, we focus on the expression optimization by changing transfection protocol and cultivation time. In the second part, we describe a robust protocol for large-scale expression and purification of membrane proteins based on affinity chromatography and gel filtration.

Keyword(s): Biochemistry (LCSH) ; Protein Science (LCSH) ; Proteins (LCSH)

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Contributing Institute(s):

1. EMBL (EMBL)
2. CSSB-EMBL (CSSB-EMBL)
3. CSSB-EMBL-CL (CSSB-EMBL-CL)

Research Program(s):

1. 899 - ohne Topic (POF4-899) (POF4-899)

Experiment(s):

1. No specific instrument

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Database coverage:
; SCOPUS

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