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Updated: Mar 3, 2026

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer
Ekaitz Errasti-Murugarren1, Arturo Rodríguez-Banqueri1,2, José Luis Vázquez-Ibar3,4
1Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology, 08028, Barcelona, Spain.
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.

