{"title":"微根头菌羧酸酯酶的克隆、重组表达和特性研究。","authors":"Michel Labuschagne","doi":"10.12688/gatesopenres.16245.1","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>The <i>Rhipicephalus microplus</i> 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.</p><p><strong>Methods: </strong>The <i>R. microplus</i> CBE gene was cloned and functionally expressed in <i>Pichia pastoris</i> following the removal of the native signal peptide. Site directed mutagenesis was used to introduce the Asp374Asn substitution.</p><p><strong>Results: </strong>Functional expression, characterization, and purification of both wild-type and mutant <i>R. microplus</i> CBE proteins was achieved using affinity chromatography under native conditions.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":12593,"journal":{"name":"Gates Open Research","volume":"8 ","pages":"87"},"PeriodicalIF":0.0000,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11813172/pdf/","citationCount":"0","resultStr":"{\"title\":\"Cloning, recombinant expression, and characterization of a <i>Rhipicephalus microplus</i> carboxylesterase.\",\"authors\":\"Michel Labuschagne\",\"doi\":\"10.12688/gatesopenres.16245.1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>The <i>Rhipicephalus microplus</i> 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.</p><p><strong>Methods: </strong>The <i>R. microplus</i> CBE gene was cloned and functionally expressed in <i>Pichia pastoris</i> following the removal of the native signal peptide. Site directed mutagenesis was used to introduce the Asp374Asn substitution.</p><p><strong>Results: </strong>Functional expression, characterization, and purification of both wild-type and mutant <i>R. microplus</i> CBE proteins was achieved using affinity chromatography under native conditions.</p><p><strong>Conclusions: </strong>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.</p>\",\"PeriodicalId\":12593,\"journal\":{\"name\":\"Gates Open Research\",\"volume\":\"8 \",\"pages\":\"87\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-08-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11813172/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Gates Open Research\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.12688/gatesopenres.16245.1\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Gates Open Research","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.12688/gatesopenres.16245.1","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/1 0:00:00","PubModel":"eCollection","JCR":"","JCRName":"","Score":null,"Total":0}
Cloning, recombinant expression, and characterization of a Rhipicephalus microplus carboxylesterase.
Background: The Rhipicephalus microplus 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 R. microplus CBE gene was cloned and functionally expressed in Pichia pastoris 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 R. microplus 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.
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