Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein.
We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.
Department of Agricultural Chemistry, Kyoto University, Japan.
A M
AM
Shimabuku
T
T
Saeki
K
K
Ueda
T
T
Komano
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D007763:Lac Operon; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases