Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro.
To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells.
Sonezaki
S
S
Department of Applied Chemistry, Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu, Japan.
Ishii
Y
Y
Okita
K
K
Sugino
T
T
Kondo
A
A
Kato
Y
Y
D018528:ATP-Binding Cassette Transporters; D049069:ATP-Dependent Proteases; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D049070:Protease La; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C053139:sulA protein, E coli; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La