Heterologous expression of cobalamin dependent class-III enzymes.

Autor: Halliwell T; Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK., Fisher K; Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK., Payne KAP; Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK; Future Biomanufacturing Research Hub (FutureBRH), Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK., Rigby SEJ; Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK., Leys D; Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. Electronic address: david.leys@manchester.ac.uk.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2021 Jan; Vol. 177, pp. 105743. Date of Electronic Publication: 2020 Aug 29.
DOI: 10.1016/j.pep.2020.105743
Abstrakt: The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation. We present a comparative study of the heterologous expression strains Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.
(Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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