Expression, purification and structural analysis of recombinant rBdh-2His6, a spermadhesin from buck (Capra hircus) seminal plasma
Antônia Sâmia F. Nascimento A , João B. Cajazeiras A , Kyria S. Nascimento A , Sara Monalisa S. Nogueira A , Bruno L. Sousa A , Edson H. Teixeira A , Luciana M. Melo B , Rodrigo Maranguape S. da Cunha A , André Luiz C. Silva A and Benildo S. Cavada A C
+ Author Affiliations
- Author Affiliations
A Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Fortaleza-CE 60440-970, Brazil.
B Laboratório de Fisiologia e Controle da Reprodução (LFCR), State University of Ceará, Faculty of Veterinary, Av. Dedé Brasil, 1700, Fortaleza-CE 60740-930, Brazil.
C Corresponding author. Email: bscavada@ufc.br
Reproduction, Fertility and Development 24(4) 580-587 https://doi.org/10.1071/RD11154
Submitted: 9 June 2011 Accepted: 3 October 2011 Published: 22 November 2011
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 1.5 mM isopropyl β-d-thiogalactoside after 2 h 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.
Additional keyword: bodhesin.
References
Bergeron, A., Villemure, M., Lazure, C., and Manjunath, P. (2005). Isolation and characterization of the major proteins of ram seminal plasma. Mol. Reprod. Dev. 71, 461–470.| Isolation and characterization of the major proteins of ram seminal plasma.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXlvFSltbo%3D&md5=d2c21e045baf273449681872f649fa35CAS | 15892046PubMed |
Cajazeiras, J. B., Melo, L. M., Albuquerque, E. S., Rádis-Baptista, G., Cavada, B. S., and Freitas, V. J. F. (2009). Analysis of protein expression and construction of prokaryotic expression system for buck (Capra hircus) spermadhesin Bdh-2 cDNA. Genet. Mol. Res. 8, 1147–1157.
| Analysis of protein expression and construction of prokaryotic expression system for buck (Capra hircus) spermadhesin Bdh-2 cDNA.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlWqu7jF&md5=8510982552eb9c8fdec05c64315314a9CAS | 19866434PubMed |
Calvete, J. J., and Sanz, L. (2007). Insights into structure–function correlations of ungulate seminal plasma potein. Soc. Reprod. Fertil. 65, 201–216.
| 1:CAS:528:DC%2BD1cXpvVyrt7w%3D&md5=6517b15cea7125530f7403c0934e5e39CAS |
Calvete, J. J., Sanz, L., Dostàlovà, Z., and Töpfer-Petersen, E. (1993). Characterization of AWN-1 glycosylated isoforms help define the zona pellucid and serine proteinase inhibitor binding region on boar spermadhesins. FESB Lett 334, 37–40.
| Characterization of AWN-1 glycosylated isoforms help define the zona pellucid and serine proteinase inhibitor binding region on boar spermadhesins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXjtFah&md5=c4c290c0e29a45a0a975bb15b7dba56eCAS |
Calvete, J. J., Sanz, L., Dostàlovà, Z., and Töpfer-Petersen, E. (1995). Spermadhesin: sperm-coating proteins involved in capacitation and zona pellucid binding. Fetility 11, 35–40.
Campanero-Rhodes, M. A., Menéndez, M., Sàiz, J. L., Sanz, L., Calvete, J. J., and Solís, D. (2006). 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. Biochemistry 45, 8227–8235.
| 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.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XlsFOqsrY%3D&md5=8eb0f5d60eef681343b4f15c3c0ca233CAS | 16819821PubMed |
Carbonell, X., and Villaverde, A. (2002). Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion. Biotechnol. Lett. 24, 1939–1944.
| Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XovVGhsb4%3D&md5=453ef53dde84d2c207c197772519d6aeCAS |
Ekhlasi-Hundrieser, M., Calvete, J. J., Von Rad, B., Hettel, C., Nimtz, M., and Töpfer-Petersen, E. (2008). Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1. Biochim. Biophys. Acta 1784, 856–862.
| 1:CAS:528:DC%2BD1cXkvVOhtbk%3D&md5=15fbcf157bd4265f2c37e0761e843dedCAS | 18359302PubMed |
Fasman, G. D. (1996). ‘Circular Dichroism and the Conformational Analysis of Biomolecules.’ (Plenum Press: New York.)
Goulding, C. W., and Perry, L. J. (2003). Protein production in Escherichia coli for structural studies by X-ray crystallography. J. Struct. Biol. 142, 133–143.
| Protein production in Escherichia coli for structural studies by X-ray crystallography.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjtVKisbY%3D&md5=fa2254c0cfa443586a4b1d3f30b2b129CAS | 12718925PubMed |
Haase, B., Schlötterer, C., Hundrieser, M. E., Kuiper, H., Distl, O., Töpfer-Petersen, E., and Leeb, T. (2005). Evolution of the spermadhesin gene family. Gene 352, 20–29.
| Evolution of the spermadhesin gene family.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXkvFWjtbg%3D&md5=666bd3a0ce2a5f8be13049803cd1d00aCAS | 15922517PubMed |
Jonasson, P., Liljeqvist, S., Nygren, P. A., and Stahl, S. (2002). Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol. Appl. Biochem. 35, 91–105.
| Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XjtFOrtrY%3D&md5=4589013f74e1d05db10ae25928d30e9dCAS | 11916451PubMed |
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
| Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXlsFags7s%3D&md5=af270e6a716a77b40e17f744b93b8b62CAS | 5432063PubMed |
Melo, L. M., Teixeira, D. I. A, Havet, A., Cunha, R. M. S, Martins, D. B. G, Castelletti, C. H. M., Souza, P. R. E., Lima-Filho, J. L., Freitas, V. J. F., Cavada, B. S., and Rádis-Baptista, G. (2008). Buck (Capra hircus) genes encode new members of the spermadhesin family. Mol. Reprod. Dev. 75, 8–16.
| 17538948PubMed |
Melo, L. M., Nascimento, A. S. F., Silveira, F. G., Cunha, R. M. S., Tavares, N. A. C., Teixeira, D. I. A., Lima-Filho, J. L., Freitas, V. J. F., Cavada, B. S., and Rádis-Baptista, G. (2009). Quantitative expression analysis of bodhesin genes in the Buck (Capra hircus) reproductive tract by real-time polymerase chain reaction (qRT-PCR). Anim. Reprod. Sci. 110, 245–255.
| Quantitative expression analysis of bodhesin genes in the Buck (Capra hircus) reproductive tract by real-time polymerase chain reaction (qRT-PCR).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsVeru73P&md5=013e0cad7d01ccd4c9d29ae4e55cd0c0CAS | 18321667PubMed |
Menéndez, M., Gasset, M., Laynez, J., López-Zumel, C., Usobiaga, P., Topfer-Petersen, E., and Calvete, J. J. (1995). Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies. Eur. J. Biochem. 234, 887–896.
| Analysis of the structural organization and thermal stability of two spermadhesins. Calorimetric, circular dichroic and Fourier-transform infrared spectroscopic studies.Crossref | GoogleScholarGoogle Scholar | 8575449PubMed |
Peti, W., and Page, R. (2007). Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr. Purif. 51, 1–10.
| Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xht1Klt7%2FF&md5=71bae9fe942ab5be98038a15dcf26f7dCAS | 16904906PubMed |
Reinert, M., Calvete, J. J., Sanz, L., Mann, K., and Töpfer-Petersen, E. (1996). Primary structure of stallion seminal plasma protein HSP-7, a zona-pellucida-binding protein of the spermadhesin family. Eur. J. Biochem. 242, 636–640.
| Primary structure of stallion seminal plasma protein HSP-7, a zona-pellucida-binding protein of the spermadhesin family.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXlsFOmtQ%3D%3D&md5=682c73e2212da09119c360cba053bcb4CAS | 9022691PubMed |
Romero, A., Varela, P., Sanz, L., Töpfer-petersen, E., and Calvette, J. J. (1996). Crystallization and preliminary X-ray diffraction analysis of boar seminal plasma spermadhesin PSP-I/PSP-II, a heterodimer of two CUB domains. FEBS Lett. 382, 15–17.
| Crystallization and preliminary X-ray diffraction analysis of boar seminal plasma spermadhesin PSP-I/PSP-II, a heterodimer of two CUB domains.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28Xhslehsrg%3D&md5=78846f35fdffe582168cd5e443ae6393CAS | 8612739PubMed |
Sørensen, H. P., and Mortensen, K. K. (2005). Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb. Cell Fact. 4, 1–8.
| Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli.Crossref | GoogleScholarGoogle Scholar | 15629064PubMed |
Sreerama, N., and Woody, R. W. (2000). Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with and expanded reference set. Anal. Biochem. 287, 252–260.
| Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with and expanded reference set.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXosFGhu7Y%3D&md5=b7dbddf849856e1e4e43cccd2214d7a2CAS | 11112271PubMed |
Tedeschi, G., Oungre, E., Mortarino, M., Negria, A., Maffeo, G., and Ronchi, S. (2000). Purification and primary structure of a new bovine spermadhesin. Eur. J. Biochem. 267, 6175–6179.
| Purification and primary structure of a new bovine spermadhesin.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M%2FjslylsQ%3D%3D&md5=13552ea281d72787318b2ca944b563f4CAS | 11012670PubMed |
Teixeira, D. I. A., Cavada, B. S., Sampaio, A. H., Havt, A., Bloch, C., , Prates, M. V., Moreno, F. B., Santos, E. A., Gadelha, C. A., Gadelha, T. S., Crisóstomo, F. S., and Freitas, V. J. S. (2002). Isolation and partial characterization of a protein from buck seminal plasma (Capra hircus) homologus to spermadhesins. Protein Pept. Lett. 9, 331–335.
| Isolation and partial characterization of a protein from buck seminal plasma (Capra hircus) homologus to spermadhesins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XltlOjurs%3D&md5=8495395ae31d186aac1003f52bc2e03bCAS |
Teixeira, D. I. A., Melo, L. M., Gadelha, C. A. A., Cunha, R. M. S., Bloch, C., , Rádis-Baptista, G., Cavada, B. S., and Freitas, V. J. F. (2006). Ion-exchange chromatography used to isolated a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma. Genet. Mol. Res. 05, 79–87.
| 1:CAS:528:DC%2BD28XltVenu7s%3D&md5=f03055ab49dd215e917855e29521f1a0CAS |
Teper, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523–533.
| Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems.Crossref | GoogleScholarGoogle Scholar |
Töpfer-Petersen, E., Romero, A., Varela, P. F., and Ekhlasi-Hundrieser, M. (1998). Spermadhesins: a new protein family. Facts, hypotheses and perspectives. Andrologia 30, 217–224.
| Spermadhesins: a new protein family. Facts, hypotheses and perspectives.Crossref | GoogleScholarGoogle Scholar | 9739418PubMed |
Töpfer-Petersen, E., Ekhlasi-Hundrieser, M., Kirchhoff, C., Leeb, T., and Sieme, H. (2005). The role of stallion seminal protein in fertilization. Anim. Reprod. Sci. 89, 159–170.
| The role of stallion seminal protein in fertilization.Crossref | GoogleScholarGoogle Scholar | 16125345PubMed |
Wempe, E., Einspanier, R., and Scheit, K. H. (1992). Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein. Biochem. Biophys. Res. Commun. 183, 232–237.
| Characterization by cDNA cloning of the mRNA of a new growth factor from bovine seminal plasma: acidic seminal fluid protein.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXhs1Wlsbc%3D&md5=9a4179e097c8ebc53eaf2e93d42debb0CAS |
Yoshizaki, L., Troncoso, M. F., Lopes, J. L., Hellman, U., Beltramini, L. M., and Wolfenstein-Todel, C. (2007). Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses. Phytochemistry 68, 2625–2634.
| Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXht1alsrjF&md5=ffbf2310524b603e6295beb734557ae0CAS | 17651769PubMed |
