Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli

To study the effect of sterols on the activity of the eukaryotic plasma mem brane transporter, the hexose-proton symporter HUP1 from the unicellular al ga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic micr oorganism containing virtually no sterols. Under certain conditions, the re combinant protein was partially active in this prokaryotic organism. The he terologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased a ctivity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevi siae membranes and solubilized, purified, and reconstituted under the same conditions as above. (C) 1999 Published by Elsevier Science B.V. All rights reserved.