Publication: One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis
Issued Date
2008-12-10
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ISSN
01410229
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2-s2.0-55949108361
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Mahidol University
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SCOPUS
Bibliographic Citation
Enzyme and Microbial Technology. Vol.43, No.7 (2008), 500-506
Suggested Citation
Kamolchanok Rukseree, Chinae Thammarongtham, Prasit Palittapongarnpim One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis. Enzyme and Microbial Technology. Vol.43, No.7 (2008), 500-506. doi:10.1016/j.enzmictec.2008.08.009 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/18800
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Title
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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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. © 2008 Elsevier Inc. All rights reserved.