title: Expression, purification and structural analysis of recombinant rBdh-2His₆, a spermadhesin from buck (Capra hircus) seminal plasma.
abstract: Spermadhesins, a family of secretory proteins from the male genital tract of ungulate species, belong to the group of animal lectins. Spermadhesins have a prominent role in different aspects of fertilisation, such as spermatozoid capacitation, acrosomal stabilisation, sperm-oviduct interaction and during sperm-oocyte fusion. Proteins (spermadhesins) in buck seminal plasma were described. In the present study, bodhesin Bdh-2 cDNA present in buck seminal plasma was subcloned with the expression plasmid pTrcHis TOPO used to transform Escherichia coli Top10 One shot cells. The recombinant clones were selected by growth in 50 µg mL⁻¹ ampicillin-containing LB broth and polymerase chain reaction amplification. Recombinant rBdh-2His₆ synthesis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by immunoblotting using monoclonal anti-His antibody. Production of rBdh-2 using low temperatures was not satisfactory. Greater production of rBdh-2 occurred with 1.5mM isopropyl βd-thiogalactoside after 2h of induction. The method used to purify rBdh-2 was affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2His₆ was evaluated by spectral profile circular dichroism (CD). The prevalence of secondary structures like β-sheets, with fewer unfolded structures and α-helices, was confirmed. The structure of rBdh-2His₆ remained stable up to 35°C. However, significant structural changes were observed at temperatures higher than 40 °C related to a distortion of the CD spectrum.
authors.affiliation: Laboratório de Moléculas Biologicamente Ativas, Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av Humberto Monte, Bloco 907, Lab 1075, Campus do Pici, Fortaleza-CE 60440-970, Brazil.
authors.forename: Antônia Sâmia F
authors.initials: AS
authors.lastname: Nascimento
authors.forename: João B
authors.initials: JB
authors.lastname: Cajazeiras
authors.forename: Kyria S
authors.initials: KS
authors.lastname: Nascimento
authors.forename: Sara Monalisa S
authors.initials: SM
authors.lastname: Nogueira
authors.forename: Bruno L
authors.initials: BL
authors.lastname: Sousa
authors.forename: Edson H
authors.initials: EH
authors.lastname: Teixeira
authors.forename: Luciana M
authors.initials: LM
authors.lastname: Melo
authors.forename: Rodrigo Maranguape S
authors.initials: RM
authors.lastname: da Cunha
authors.forename: André Luiz C
authors.initials: AL
authors.lastname: Silva
authors.forename: Benildo S
authors.initials: BS
authors.lastname: Cavada
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D006041:Goats / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D037102:Lectins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008297:Male; D014176:Protein Biosynthesis / Q000187:drug effects; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012661:Semen / Q000378:metabolism*; D029589:Seminal Plasma Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
chemical_list: D037102:Lectins; D011994:Recombinant Proteins; D029589:Seminal Plasma Proteins; C425042:spermadhesin; D007544:Isopropyl Thiogalactoside