Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain.
A rapid and efficient method has been used for the purification of a Plantago major sucrose carrier from Saccharomyces cerevisiae. The C-terminal fusion of a bacterial biotin acceptor domain to the carrier protein did not interfere with the targeting to the yeast plasma membrane nor with the catalytic activity of the sucrose carrier. The chimeric construct is biotinylated by yeast cells in vivo and represents the only biotinylated protein in yeast membranes. Solubilized biotinylated carrier protein binds selectively to immobilized monomeric avidin and can be eluted as pure protein with free biotin. The purified protein is functionally active and catalyzes the energy-dependent transport of sucrose into proteoliposomes.
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Germany.
J
J
Stolz
B
B
Darnhofer-Demar
N
N
Sauer
D000595:Amino Acid Sequence; D001360:Avidin / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005822:Genetic Vectors; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010943:Plantago / Q000737:chemistry* / Q000235:genetics; D010946:Plants, Medicinal; D011510:Proteolipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C020485:proteoliposomes; C078729:sucrose transport protein, plant; D001360:Avidin; D001710:Biotin