PMID:3130379
| Citation |
Hanada, K, Yamato, I and Anraku, Y (1988) Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein. J. Biol. Chem. 263:7181-5 |
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| 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. |
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| Keywords |
Amino Acid Transport Systems, Neutral; Carrier Proteins/genetics; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Cloning, Molecular; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Genes; Genes, Bacterial; Kinetics; Liposomes; Molecular Weight; Proline/metabolism; Proteolipids/metabolism; Recombinant Fusion Proteins/isolation & purification; Recombinant Fusion Proteins/metabolism; Recombinant Proteins/metabolism; Symporters; beta-Galactosidase/genetics |
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