Recombinant expression and affinity purification of a novel epididymal human sperm-binding protein, BSPH1.
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) Escherichia coli cells. Similar to other BSP proteins, BSPH1 contains two fibronectin type-II (Fn2) domains, each consisting of two 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-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 His(6)-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 orthologue 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 to elucidate the role of BSPH1 in human sperm functions and fertility.
Lefebvre
Jasmine
J
Research Centre, Maisonneuve-Rosemont Hospital, University of Montreal, Quebec, Canada.
Boileau
Guy
G
Manjunath
Puttaswamy
P
D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D004530:Egg Yolk / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008297:Male; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029607:Seminal Vesicle Secretory Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism
C519889:BSPH1 protein, human; D008077:Lipoproteins, LDL; D008081:Liposomes; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D029607:Seminal Vesicle Secretory Proteins