Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

FEBS Letters

Sauer, Norbert¹  Stolz, Jürgen¹  Darnhofer-Demar, Brigitte¹ 

[1] Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, D-93040 Regensburg, Germany

关键词: Biotinylation;    Heterologous expression;    Protein purification;    Reconstitution;    Sucrose transporter;    CCCP;    carbonyl cyanide m-chlorophenyl hydrazone;    cytochrome-c-oxidase;    ferrocytochrome c:oxygen oxidoreductase (EC 1.9.3.1);    OG;    octyl-β-d-glucoside;    TMPD;    N;    N;    N′;    N′-tetrametthyl-p-phenylenediamine;   

DOI  :  10.1016/0014-5793(95)01333-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown