title: Small-Scale Screening to Large-Scale Over-Expression of Human Membrane Proteins for Structural Studies.
abstract: Membrane protein structural studies are frequently hampered by poor expression. The low natural abundance of these proteins implies a need for utilizing different heterologous expression systems. E. coli and yeast are commonly used expression systems due to rapid cell growth at high cell density, economical production, and ease of manipulation. Here we report a simplified, systematically developed robust strategy from small-scale screening to large-scale over-expression of human integral membrane proteins in the mammalian expression system for structural studies. This methodology streamlines small-scale screening of several different constructs utilizing fluorescence size-exclusion chromatography (FSEC) towards optimization of buffer, additives, and detergents for achieving stability and homogeneity. This is followed by the generation of stable clonal cell lines expressing desired constructs, and lastly large-scale expression for crystallization. These techniques are designed to rapidly advance the structural studies of eukaryotic integral membrane proteins including that of human membrane proteins.
authors.affiliation: Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India. sarika.chaudhary@igib.in.
authors.forename: Sarika
authors.initials: S
authors.lastname: Chaudhary
authors.affiliation: Systems Biology Group, CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India.
authors.forename: Sukanya
authors.initials: S
authors.lastname: Saha
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, 94158, USA.
authors.forename: Sobrahani
authors.initials: S
authors.lastname: Thamminana
authors.affiliation: Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, 94158, USA. stroud@msg.ucsf.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics
chemical_list: D008565:Membrane Proteins
keywords: FSEC; Large-scale expression; Mammalian cell culture; Membrane protein expression