Purification of two active fusion proteins of the Na+‐dependent citrate carrier of Klebsiella pneumoniae

FEBS Letters

Dimroth, Peter¹  Bott, Michael¹  Pos, Klaas M.¹ 

[1] Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland

关键词: citS gene;    Secondary active transport;    Citrate carrier;    Sodium ion/citrate symport;    Fusion proteins;    Affinity chromatography;    Klebsiella pneumoniae;    CitS;    sodium-dependent citrate carrier;    CitSα;    CitS with a C-terminally attached biotinylation domain derived from the α-subunit of oxaloacetate decarboxylase;    CitSHis;    CitS with an N-terminally attached polyhistidine-tail;    FCCP;    carbonyl cyanide p-trifluoromethoxyphenylhydrazone;    IPTG;    isopropyl-β-d-thiogalactopyranoside oadGAB;    genes encoding the γ-;    α-;    and β-subunit of oxaloacetate decarboxylase from Klebsielia pneumoniae;    respectively;   

DOI  :  10.1016/0014-5793(94)00502-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The sodium-ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) was purified by means of bioengineerical methods. By fusing the biotin acceptor domain of the α-subunit of the oxaloacetate decarboxylase of K pneumoniae to the C-terminus of CitS, purification of the carrier was achieved by use of a monomeric avidin-Sepharose column. Additionally, we were able to purify a CitS-protein with an N-terminal histidine-tag by immobilized metal chelate affinity chromatography (with Ni²⁺-nitrilotriacetic acid-(NTA-) resin). Both purified fusion proteins showed citrate transport activity after reconstitution into liposomes by the freeze/thaw/sonication procedure.

【 授权许可】

Unknown