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Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter.
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- Additional Information
- Source:
Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE
- Publication Information:
Publication: Totowa, NJ : Humana Press
Original Publication: Clifton, N.J. : Humana Press,
- Subject Terms:
- Abstract:
Obtaining enough quantity of recombinant membrane transport proteins with optimal purity and stability for structural studies is a remarkable challenge. In this chapter, we describe a protocol to engineer SteT, the amino acid transporter of Bacillus subtilis, in order to improve its heterologous expression in Escherichia coli and its stability in detergent micelles. We built a library of 70 SteT mutants, combining a random mutagenesis protocol with a split GFP assay as reporter of protein folding and membrane insertion. Mutagenesis was restricted to residues situated in the transmembrane domains. Improved versions of SteT were successfully identified after analyzing the expression yield and monodispersity in detergent micelles of the library's members.
- Contributed Indexing:
Keywords: FSEC; Heterologous expression; LAT; Membrane transport proteins; Split GFP; SteT
- Accession Number:
0 (Amino Acid Transport Systems)
0 (Bacterial Proteins)
0 (Detergents)
0 (Recombinant Fusion Proteins)
147336-22-9 (Green Fluorescent Proteins)
- Publication Date:
Date Created: 20170505 Date Completed: 20180220 Latest Revision: 20201209
- Publication Date:
20240829
- Accession Number:
10.1007/978-1-4939-6887-9_11
- Accession Number:
28470605
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