Cloning, recombinant expression, and characterization of a Rhipicephalus microplus carboxylesterase.

<ns3:p>Background The <ns3:italic>Rhipicephalus microplus</ns3:italic> carboxylesterase (CBE) is involved in synthetic pyrethroid (SP) hydrolysis and historic evidence suggests that a non-synonymous mutation (Asp374Asn) in CBE is associated with increased resistance towards SP-based acaricides. Functional expression and characterization of the wild-type and mutant CBE is required to understand the impact of the mutation on SP-based resistance. Methods The <ns3:italic>R. microplus</ns3:italic> CBE gene was cloned and functionally expressed in <ns3:italic>Pichia pastoris</ns3:italic> following the removal of the native signal peptide. Site directed mutagenesis was used to introduce the Asp374Asn substitution. Results Functional expression, characterization, and purification of both wild-type and mutant <ns3:italic>R. microplus</ns3:italic> CBE proteins was achieved using affinity chromatography under native conditions. Conclusions This report provides the necessary information for the tick research community to produce recombinant tick derived CBE proteins and to characterize the recombinant proteins towards substrates of interest.</ns3:p>