Data_Sheet_1_Functional Expression and Characterization of Tetrachloroethene Dehalogenase From Geobacter sp..PDF (378.11 kB)

Data_Sheet_1_Functional Expression and Characterization of Tetrachloroethene Dehalogenase From Geobacter sp..PDF

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posted on 10.08.2018, 04:05 by Ryuki Nakamura, Tomohiro Obata, Ryota Nojima, Yohey Hashimoto, Keiichi Noguchi, Takahiro Ogawa, Masafumi Yohda

Reductive dehalogenase (RDase) consists of two parts, RdhA and RdhB. RdhA is the catalytic subunit, harboring a cobalamin cofactor and two Fe–S clusters. RdhA is anchored to the cytoplasmic membrane via the membrane anchoring subunit, RdhB. There are many genes encoding RDases in the genome of organohalide-respiring bacteria, including Dehalococcoides spp. However, most genes have not been functionally characterized. Biochemical studies on RDases have been hampered by difficulties encountered in their expression and purification. In this study, we have expressed, purified and characterized RdhA of RDase for tetrachloroethene (PceA) from Geobacter sp. PceA was expressed as a fusion protein with a trigger factor tag in Escherichia coli. PceA was purified and denatured in aerobic condition. Subsequently, this protein was refolded in the presence of FeCl3, Na2S and cobalamin in anaerobic condition. The reconstituted PceA exhibited dechlorination ability for tetrachloroethene. UV-Vis spectroscopy has shown that it contains cobalamin and Fe-S clusters. Since this method requires anaerobic manipulation only in the reconstituting process and has a relatively high yield, it will enable further biochemical studies of RDases.

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