High level of soluble expression in Escherichia coli and characterisation of the CyaA pore-forming fragment from a Bordetella pertussis Thai clinical isolate |
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Authors: | Busaba Powthongchin Chanan Angsuthanasombat |
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Affiliation: | (1) Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand;(2) Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakornpathom, 73000, Thailand |
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Abstract: | Bordetella pertussis adenylate cyclase toxin-haemolysin (CyaA) can permeabilise erythrocytes by forming lytic pores. Here, a gene segment encoding CyaA pore-forming (CyaA-PF) domain cloned from genomic DNA of B. pertussis Thai isolate was over-expressed in Escherichia coli as a 126-kDa soluble protein which cross-reacted with anti-RTX monoclonal antibody. By co-expressing with acyltransferase CyaC, the CyaA-PF protein was found palmitoylated at Lys983. Unlike E. coli lysate with the non-acylated form, the lysate containing acylated CyaA-PF exhibited high haemolytic activity against sheep erythrocytes. This study presents that the recombinant CyaA-PF protein comprising pore-forming domain can be expressed separately as soluble native-folded precursor that conserves at least part of its functionality. |
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Keywords: | Adenylate cyclase toxin Acyltransferase Bordetella pertussis Palmitoylation Pore-forming toxin Sheep erythrocyte |
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