title: Employing Escherichia coli to functionally express, purify, and characterize a human transporter.
abstract: Large-scale purification of recombinant human membrane proteins represents a rate-limiting step toward the understanding of their role in health and disease. There are only four mammalian membrane proteins of known structure, and these were isolated from natural sources (see http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html). In addition, genetic diseases of membrane proteins are frequently caused by trafficking defects, and it is enigmatic whether these mutants are functional. Here, we report the employment of Escherichia coli for the functional expression, purification, and reconstitution of a human membrane protein, the human Na+/glucose cotransporter (hSGLT1). The use of an E. coli mutant defective in the outer membrane protease OmpT, incubation temperatures below 20 degrees C, and transcriptional regulation from the lac promoter/operator are crucial to reduce proteolytic degradation. Purification of a recombinant hSGLT1 through affinity chromatography yields about 1 mg of purified recombinant hSGLT1 per 3 liters of cultured bacterial cells. Kinetic analysis of hSGLT1 in proteoliposomes reveals that a purified recombinant transporter, which is missorted in eukaryotic cells, retains full catalytic activity. These results indicate the power of bacteria to manufacture and isolate human membrane proteins implicated in genetic diseases.
authors.affiliation: Department of Physiology, School of Medicine, University of California, Los Angeles, CA 90095-1751, USA. mquick@mednet.ucla.edu
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Ernest M
authors.initials: EM
authors.lastname: Wright
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051269:Sodium-Glucose Transporter 1
chemical_list: D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D049452:Green Fluorescent Proteins