title: Purification and antigenicity of two recombinant forms of Boophilus microplus yolk pro-cathepsin expressed in inclusion bodies.
abstract: The tick Boophilus microplus is a bovine ectoparasite present in tropical and subtropical areas of the world and the use of vaccines is a promising method for tick control. BYC is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus and was proposed as an important antigen in the development of an anti-tick vaccine. The cDNA of BYC was amplified by PCR and cloned for expression in two forms with and without thioredoxin fusion protein (Trx), coding recombinant proteins named rBYC-Trx and rBYC, respectively. Expression, solubility, and yields of the two forms were analyzed. The recombinant proteins were expressed in inclusion bodies (IBs) and three denaturant agents (N-lauroyl sarcosine, guanidine hydrochloride, and urea) were tested for IBs solubilization. The N-lauroyl sarcosine solubilized 90.4 and 92.4% of rBYC-Trx and rBYC IBs, respectively, and was the most efficient denaturant. Two recombinant forms were affinity-purified by Ni2+-Sepharose under denaturing conditions. After dialysis, the yield of soluble protein was 84.1% for r-BYC-Trx and 5.9% for rBYC. These proteins were immune-reactive against sera from rabbit, mouse, and bovine previously immunized with native BYC, which confirms the antigenicity of the recombinant BYCs expressed in the Escherichia coli system.
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Porto Alegre-RS 91501-970, Brazil.
authors.forename: Alexandre T
authors.initials: AT
authors.lastname: Leal
authors.forename: Paula C
authors.initials: PC
authors.lastname: Pohl
authors.forename: Carlos A S
authors.initials: CA
authors.lastname: Ferreira
authors.forename: Maria C L
authors.initials: MC
authors.lastname: Nascimento-Silva
authors.forename: Marcos H F
authors.initials: MH
authors.lastname: Sorgine
authors.forename: Carlos
authors.initials: C
authors.lastname: Logullo
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Oliveira
authors.forename: Sandra E
authors.initials: SE
authors.lastname: Farias
authors.forename: Itabajara
authors.initials: I
authors.lastname: da Silva Vaz
authors.forename: Aoi
authors.initials: A
authors.lastname: Masuda
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000939:Epitopes; D005260:Female; D005786:Gene Expression Regulation; D019791:Guanidine / Q000494:pharmacology; D002479:Inclusion Bodies / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012521:Sarcosine / Q000494:pharmacology; D012995:Solubility; D013987:Ticks / Q000235:genetics*; D014508:Urea / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D000939:Epitopes; D011994:Recombinant Proteins; D014508:Urea; D016282:Aspartic Acid Endopeptidases; C113293:Boophilus yolk pro-cathepsin; D019791:Guanidine; D012521:Sarcosine