Expression, purification and structural analysis of recombinant rBdh-2His₆, a spermadhesin from buck (Capra hircus) seminal plasma.

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.

[1]  L. M. Melo,et al.  Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA. , 2009, Genetics and molecular research : GMR.

[2]  J. Calvete,et al.  Spermadhesins: A new protein family. Facts, hypotheses and perspectives , 2009, Andrologia.

[3]  D. Teixeira,et al.  Quantitative expression analysis of Bodhesin genes in the buck (Capra hircus) reproductive tract by real-time polymerase chain reaction (qRT-PCR). , 2009, Animal reproduction science.

[4]  J. Calvete,et al.  Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1. , 2008, Biochimica et biophysica acta.

[5]  D. Teixeira,et al.  Buck (Capra hircus) genes encode new members of the spermadhesin family , 2008, Molecular reproduction and development.

[6]  M. F. Troncoso,et al.  Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses. , 2007, Phytochemistry.

[7]  J. Calvete,et al.  Zinc ions induce the unfolding and self-association of boar spermadhesin PSP-I, a protein with a single CUB domain architecture, and promote its binding to heparin. , 2006, Biochemistry.

[8]  T. Leeb,et al.  The role of stallion seminal proteins in fertilisation. , 2005, Animal reproduction science.

[9]  C. Lazure,et al.  Isolation and characterization of the major proteins of ram seminal plasma , 2005, Molecular reproduction and development.

[10]  C. Schlötterer,et al.  Evolution of the spermadhesin gene family. , 2005, Gene.

[11]  H. P. Sørensen,et al.  Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli , 2005 .

[12]  L. J. Perry,et al.  Protein production in Escherichia coli for structural studies by X-ray crystallography. , 2003, Journal of structural biology.

[13]  Antonio Villaverde,et al.  Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion , 2002, Biotechnology Letters.

[14]  D. Teixeira,et al.  Isolation and partial characterisation of a protein from buck seminal plasma (Capra hircus), homologous to spermadhesins. , 2002, Protein and peptide letters.

[15]  Per Jonasson,et al.  Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli , 2002, Biotechnology and applied biochemistry.

[16]  N. Sreerama,et al.  Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. , 2000, Analytical biochemistry.

[17]  G. Tedeschi,et al.  Purification and primary structure of a new bovine spermadhesin. , 2000, European journal of biochemistry.

[18]  J. Calvete,et al.  Primary structure of stallion seminal plasma protein HSP-7, a zona-pellucida-binding protein of the spermadhesin family. , 1996, European journal of biochemistry.

[19]  J. Calvete,et al.  Crystallization and preliminary X‐ray diffraction analysis of boar seminal plasma spermadhesin PSP‐I/PSP‐II, a heterodimer of two CUB domains , 1996, FEBS letters.

[20]  M. Gasset,et al.  Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies. , 1995, European journal of biochemistry.

[21]  J. Calvete,et al.  Characterization of AWN‐1 glycosylated isoforms helps define the zona pellucida and serine proteinase inhibitor‐binding region on boar spermadhesins , 1993, FEBS letters.

[22]  R. Einspanier,et al.  Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein. , 1992, Biochemical and biophysical research communications.

[23]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[24]  Rebecca Page,et al.  Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. , 2007, Protein expression and purification.

[25]  K. Terpe.  Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems , 2002, Applied Microbiology and Biotechnology.