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Mol. Hum. Reprod. Advance Access published online on December 17, 2008
Molecular Human Reproduction, doi:10.1093/molehr/gan077
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Recombinant expression and affinity purification of a novel epididymal human sperm-binding protein, BSPH1

Jasmine Lefebvre1,2, Guy Boileau2 and Puttaswamy Manjunath1,3,4

1Research Centre, Maisonneuve-Rosemont Hospital, University of Montreal, Montreal, Quebec, H1T 2M4 Canada 2Department of Biochemistry, University of Montreal, Montreal, Quebec, H1T 2M4 Canada 3Department of Medicine, University of Montreal, Montreal, Quebec, H1T 2M4 Canada

4 To whom correspondence should be addressed at: Centre de Recherche, Hôpital Maisonneuve-Rosemont, 5415 boulevard de l'Assomption, Montréal, Québec, H1T 2M4, Canada. Tel.: +1 514 252 3562 or +1 514 252 3400 (ext. 3329). Fax: +1 514 252 3430 E-mail address: puttaswamy.manjunath{at}umontreal.ca

Mammalian sperm undergo a series of maturation steps before acquiring fertilization competence. Our previous work demonstrated the importance of Binder of SPerm (BSP) proteins in bovine sperm capacitation. Recent studies identified a BSP-homologous DNA sequence in the human genome (BSPH1) and mRNA expression in the epididymis. The aim of this study was to develop an efficient method to express and purify recombinant human BSPH1. BSPH1 accumulates in inclusion bodies when expressed with an N-terminal hexahistidine tag in BL21(DE3) E.coli cells. Similar to other BSP proteins, BSPH1 contains 2 fibronectin type-II (Fn2) domains, each consisting of 2 disulfide bonds. Therefore, when expressed in Origami B(DE3)pLysS cells, a strain favouring disulfide bond formation, an improvement in soluble protein yield was observed. However, protein was aggregated, which complicated subsequent purification steps. Expression of glutathione-S-transferase (GST)-tagged BSPH1 in both cell types also led to accumulation in inclusion bodies. Finally, successful production of soluble and active protein was achieved when BSPH1 was expressed as a His6-thioredoxin-tagged protein. Recombinant protein bound phosphatidylcholine liposomes, low-density lipoproteins and human sperm, therefore displayed binding activities common to all BSP-family proteins, which may indicate similar biological function(s). This approach was also successful in producing the murine ortholog of BSPH1 in the soluble and active form. Thus, fusion to thioredoxin and expression in Origami B(DE3)pLysS cells may constitute a strategy applicable to all BSP-family proteins, and possibly to other proteins containing Fn2 domains. This work is important in order to elucidate the role of BSPH1 in human sperm functions and fertility.

Key Words: epididymal protein/binder of sperm(BSP) proteins/fibronectin(Fn)2 domains/recombinant protein expression/thioredoxin

Submitted on August 8, 2008; resubmitted on November 25, 2008; accepted on December 3, 2008.


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