Overproduction and purification of Lon protease from Escherichia coli using a maltose-binding protein fusion system.
Lon protease, which plays a major role in degradation of abnormal proteins in Escherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form in E. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-L-phenylalanyl-L-leucyl-phenylalanyl-beta-D-methoxynaphthyl amide) and protein (alpha-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes.
Sonezaki
S
S
Department of Applied Chemistry, Faculty of Engineering, Kyusyu Institute of Technology, Kitakyusyu, Japan.
Kondo
A
A
Oba
T
T
Ishii
Y
Y
Kato
Y
Y
Nakayama
H
H
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D018528:ATP-Binding Cassette Transporters; D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D001709:Biotechnology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002364:Caseins; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D049070:Protease La; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013489:Suppression, Genetic
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002364:Caseins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C067047:succinyl-phenylalanyl-leucyl-phenylalanine-4-methoxynaphthylamide; D008320:Maltose; D015081:2-Naphthylamine; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La