The conserved motor domain of Aspergillus fumigatus BimC kinesin (EG02) has been produced and purified from a prokaryotic expression system. The recombinant protein contains six histidine residues at the amino terminus (His-tag)and has an approximate molecular weight of 50 kDa. A. fumigatus BimC has been determined to be biologically active in a microtubule-activated ATPase activity test. The protein is supplied as a white lyophilized powder.
Protein purity is determined by scanning densitometry of Coomassie stained protein on a 12% gel. His-BimC protein was determined to be >95% pure (see Figure 1).
Figure 1. His-BimC Kinesin Motor Domain protein purity gel. A 10 µg sample of recombinant His-BimC protein (approx. 50 kDa) was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie blue. Protein quantitation was determined using Advanced Protein assay (Cat. # ADV02). Mark12 molecular weight markers are from Invitrogen.
Biological Activity - Microtubule Activated ATPase Assay
BimC ATPase activity was measured by monitoring real time free phosphate generation using the Kinesin ELIPA Assay Kit (Cat. # BK060). The assay is based upon an absorbance shift (330 nm to 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6mercapto-7-methylpurine in the presence of inorganic phosphate (Pi). One molecule of Pi will yield one molecule of 2-amino-6mercapto-7-methylpurine in an essentially irreversible reaction. Hence, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the kinesin ATPase reaction. Under the conditions outlined below, the Vmax for BimC microtubuleactivated ATPase activity has a minimum activity of 250 nmoles ATP generated per minute per mg of protein (Figure 2).
Figure 2. EG02 microtubule-activated ATPase activity. Recombinant BimC from A. fumigatus was assayed for microtubuleactivated ATPase activity in triplicate along with human recombinant Eg5 (Cat. # EG01) according to the method described. Control reactions were carried out in the absence of motor protein (microtubules only) and in the absence of microtubules (data not shown).
|Funk, C. Joel et al.||Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators||Analytical Biochemistry||2004||ISSN 0003-2697|
|Wada, Yuuko et al.||Evidence for a Novel Affinity Mechanism of Motor-assisted Transport Along Microtubules||Molecular Biology of the Cell||2000||ISSN 1059-1524|
|Endow, Sharyn A. et al.||Determinants of Kinesin Motor Polarity||Science||1998||ISSN 0036-8075|
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