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K. Hanada, I. Yamato & Y. Anraku 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 β-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-β-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 β-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 μM and 31 μM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.
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