Purification of two active fusion proteins of the Na(+)-dependent citrate carrier of Klebsiella pneumoniae.
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 alpha-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.
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Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, Zürich, Switzerland.
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D001426:Bacterial Proteins; D001483:Base Sequence; D001692:Biological Transport; D001710:Biotin; D002262:Carboxy-Lyases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002951:Citrates / Q000378:metabolism*; D019343:Citric Acid; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011510:Proteolipids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012964:Sodium / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; C086246:CitS protein, bacteria; D002951:Citrates; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C020485:proteoliposomes; D019343:Citric Acid; D001710:Biotin; D012964:Sodium; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase