Production and proteolytic assay of lethal factor from Bacillus anthracis.
Bacillus anthracis is the causative agent of anthrax. The major virulence factors are a poly-D-glutamic acid capsule and three-protein component exotoxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa), respectively. These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin or edema toxin), causing different pathogenic responses in animals and cultured cells. In this study, we constructed and produced rLF as a form of GST fusion protein in Escherichia coli. rLF was rapidly purified through a single affinity purification step to near homogeneity. Furthermore, we developed an in vitro immobilized proteolytic assay of LF under the condition containing full-length native substrate, MEK1, rather than short synthetic peptide. The availability of full-length substrate and of an immobilized LF assay could facilitate not only the in-depth investigation of structure-function relationship of the enzyme toward its substrate but also wide spectrum screening of inhibitor collections based on the 96-well plate system.
Kim
Joungmok
J
Department of Chemistry, Hanyang University, 133-791 Seoul, Republic of Korea.
Kim
Young-Myung
YM
Koo
Bon-Sung
BS
Chae
Young-Kyu
YK
Yoon
Moon-Young
MY
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D011994:Recombinant Proteins; C030325:anthrax toxin