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One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis

Paper ID Volume ID Publish Year Pages File Format Full-Text
17657 42687 2008 7 PDF Available
Title
One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis
Abstract

Rv0363c (fba), encoding Class II fructose-bisphosphate aldolase (FBA), is one of the potential drug targets identified in our laboratory based on minimal gene set concept. The wild-type enzyme overproduction in E. coli had been reported. However, the purification procedure was relatively tedious and the yield was low. In this study, five histidine codons were introduced into the 3′ end of the amplified fba fragments. The expressed C-terminal histidine-tagged Class II FBA was produced in E. coli BL21 (DE3) and easily purified using immobilized metal affinity chromatography. The purified his-tagged FBA was characterized. Its biochemical properties were compared to the non-his-tagged enzyme purified according to the previous report. Both FBAs have similar characteristics such as native/subunit molecular mass, kinetic parameters, and temperature/pH optima and stability. The C-terminal his-tagged FBA can be a surrogate for the native enzyme and used for screening of inhibitors of FBA. This developed expression system will pave the way for high-throughput screening and crystallization studies. Moreover, in this study, a colorimetric FBA assay has been simplified to facilitate the mass screening of inhibitor of FBA.

Keywords
Fructose-1,6-bisphosphate aldolase; Mycobacterium tuberculosis; Aldolase assay; Immobilized metal affinity chromatography
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One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 43, Issue 7, 10 December 2008, Pages 500–506
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us