Functional purification of a bacterial ATP-binding cassette transporter protein (MalK) from the cytoplasmic fraction of an overproducing strain.
The malK gene of Salmonella typhimurium encoding the ATP-hydrolyzing subunit of the ATP-Binding Cassette (ABC) transporter for maltose was subcloned into the pRSET5d expression vector. Subsequently, the resulting plasmid (pES67) was introduced into Escherichia coli strain BL21(DE3)/pLysS. When strain BL21-(DE3)/pLysS/pES67 was grown at 30 degrees C in a tryptone-phosphate medium (J.T. Moore, A. Uppal, F. Maley, and G. F. Maley, Protein Expression Purif. 4, 160-163, 1993), the addition of isopropyl beta-thiogalactoside resulted in the synthesis of large amounts of MalK protein. After cell disrupture about 60% of MalK was recovered with the soluble (cytoplasmic) fraction. The protein was purified by ion exchange chromatography and dye ligand affinity chromatography. The purified MalK protein displayed enzymatic properties similar to those of a preparation purified and renatured from inclusion bodies (S. Morbach, S. Tebbe, and E. Schneider, J. Biol. Chem. 268, 18617-18621, 1993). Thus, our results disprove the view that the biochemical properties of a protein renatured from inclusion bodies might be artefactual. In addition, we provide further evidence that the modification of growth conditions and the use of a T7 expression system can be a useful approach to overcome at least in part the formation of inclusion bodies.
Schneider
E
E
Arbeitsgruppe Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Germany.
Linde
M
M
Tebbe
S
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; C489399:MalK protein, Salmonella typhimurium; D008320:Maltose; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases