title: Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein.
abstract: We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y. (1987) J. Biol. Chem. 262, 14100-14104). The proline carrier was purified from this site specifically cleavable fusion protein. Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography. The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety. The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography. In this way, the proline carrier was purified to more than 95% homogeneity of the protein. Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.
mesh_terms: D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D011392:Proline / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D027981:Symporters; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D027981:Symporters; C041832:proline transport protein; C020485:proteoliposomes; C060609:PutP protein, E coli; D011392:Proline; D001616:beta-Galactosidase