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Onchocerca volvulus chitinase (OvCHT1) |
 | | | | Onchocerca volvulus chitinase (OvCHT1) | | | |
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Description:
Onchocerca volvulus chitinase (OvCHT1) is expressed during development in the black fly vector in the infective L3 larvae and then secreted during post-infective development in the human host (1, 2). The specific role of the chitinase is not yet clear.
Source:
Isolated from the supernatant of Spodoptera frugiperda (Sf9) insect cells infected with an AcNPV chiA minus recombinant baculovirus carrying the OvCHT1 gene cloned from Onchocerca volvulus.
AcNPV (Autographa californica nuclear polyhedrosis virus)
Enzyme Properties
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 53 kDa and Apparent: 65 kDa
Reaction & Storage Conditions
Unit Definition:
One unit is defined as the amount of enzyme required to release the equivalent fluorescence produced by 1 pmol 4-methylumbelliferone from the substrate 4-methylumbelliferyl-N,N',N"-triacetyl-β-chitotrioside in a total reaction volume of 100 μl in 1 minute at 25°C in 200 mM NaCl with 20 mM NaPO4 (pH 6.0), 1 mM EDTA and 20 µM 4-methylumbelliferyl-N,N',N"-triacetyl-β-chitotrioside.
Concentration:
10,000 units/ml
Storage Conditions:
20 mM Tris-HCl
200 mM NaCl
1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Assay Conditions::
0.2 M NaCl, 20 mM NaPO4 (pH 6.0), 1 mM EDTA, 20 µM 4-methylumbelliferyl-N,N',N''-triacetyl-β-chitotrioside
Chitin Binding Assay::
100 units of OvCHT1 were added to 100 µl of chitin resin in binding buffer (200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA) mixed and left on ice for 15 minutes. After centrifugation to settle the resin, the supernatant was collected containing the unbound protein sample. The resin was then washed twice with 1 ml of binding buffer, centrifuged, and the wash samples collected. The chitinase activity of the initial load, the unbound sample, and the wash samples were determined. Protein samples of the load, unbound protein, wash and resin were also run on an SDS-PAGE gel.
Results: No chitinase activity was detected in the unbound or wash samples. Also, SDS-PAGE gel shows that the 65 kDa band corresponding to the OvCHT1 was not present in the unbound or wash samples but was present in the chitin resin sample. Therefore, the OvCHT1 was bound to the chitin resin.
[3H]-chitin Assay::
160 units of OvCHT1 were incubated with 10,000 cpm [3H]-chitin in 200 mM NaCl, 20 mM NaPO4 (pH 6.0), 1 mM EDTA and 500 µg/ml BSA in 200 µl at 37°C. Aliquots were removed at various time points up to 24 hours, mixed with unlabelled chitin, and after centrifugation, the total soluble cpm for each time point was determined.
Results: 160 units of OvCHT1 released 59% of the total soluble cpm in 24 hours.
References
- Wu, Y. et al. (2001) J. Biol. Chem., 276, 42557-42564.
- Wu, Y. et al. (1996) Mol. Biochem. Parasitol., 75, 207-219.
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