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.de͞michel͞ public͞memprotstruct.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°C, and transcriptional regulation from the lac promoter͞operator 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.I ntegral membrane proteins such as channels and transporters are essential for all living organisms by mediating the passage of ions and molecules across membranes. The understanding of how membrane proteins work in health and disease has been restricted largely because of the lack of purified protein. Most membrane proteins are present at low concentrations in their native tissue, and their overexpression is a prerequisite for purification. The use of eukaryotic expression systems has illuminated mechanistic features of human membrane proteins, but these are unsuitable for their large-scale purification (1, 2). In addition, genetic disorders of membrane proteins are frequently caused by improper trafficking in cells, and it is not known whether missorted proteins retain their catalytic activity (3). To obtain structural information on membrane proteins, prokaryotic homologues have been most amenable because they can often be expressed in bacteria in large quantities (4). However, the inexpensive and less time-consuming overexpression of animal and human integral proteins of the plasma membrane has not been applicable, and there are only a few examples in which eukaryote membrane proteins are functional when expressed in prokaryotic cells (5-11). Here, we report the employment of Escherichia coli for the expression, purification, and reconstitution of a fully functional recombinant human sodium͞glucose transporter (hSGLT1)-a central protein for the homeostasis of glucose, salt, and water (12-14). Our strategy is based in part on our group's previous success in purifying functional transporters from bacteria (15, 16). Experimental ProceduresGenetic Engineering. A casset...

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpT

“…The cleavage of sequences containing dibasic residues has been shown to be important for the inactivation of antibiotic peptides and colicins, the proteolysis of bacterial membrane proteins in trans, and the degradation of recombinant proteins expressed in E. coli (2,4,11,12,20,25,26,29,30). Since the enzyme is a membrane protein, it fractionates with the insoluble fraction in cell lysates and copurifies with protein inclusion bodies (33).…”

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

“…13 Although functional characterization of all test proteins is beyond the scope of this report, LldP-GpA-GFP was partially characterized to demonstrate the feasibility of generating functional protein using the GpA-GFP-fusion. The plasmid construct expressing LldP-GpA-GFPHis 8 was transformed into the knockout E. coli strain ECL5106.…”

“…2,13 Expanding upon these early results, we engineered a set of vectors for monitoring expression, detergent screening and stability testing of both C in and C out proteins with GFP fluorescence. We have termed these vectors pWarf(À) and pWarf(þ) [ Fig.…”

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

“…As rare examples of bacterial transporters have been reported to function when expressed in Xenopus oocytes (Quick et al 2002), Rv1739c was subcloned into the oocyte transcription vector pXT7 behind the Kozak sequence from Kcc1/Slc12a1, and functionally tested in Xenopus oocytes for 35 S-sulfate influx and efflux. The 30 min sulfate uptake into individual oocytes from ND-96 medium at pH 6.0 was slightly increased over background (0.23±0.01 pmol min −1 , n=30) by Rv1739c expression in oocytes injected with 10 ng RNA (0.31±0.02 pmol min −1 , n=48, p<0.005), but not at pH 7.4 (n=30).…”