High level of soluble expression in Escherichia coli and characterisation of the CyaA pore-forming fragment from a Bordetella pertussis Thai clinical isolate.
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 Lys(983). 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.
Powthongchin
Busaba
B
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand.
Angsuthanasombat
Chanan
C
D037361:Adenylate Cyclase Toxin / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D001886:Bordetella pertussis / Q000235:genetics* / Q000302:isolation & purification; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006461:Hemolysis; D006801:Humans; D054878:Lipoylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D012756:Sheep; D013785:Thailand; D014917:Whooping Cough / Q000382:microbiology
D037361:Adenylate Cyclase Toxin; D004269:DNA, Bacterial