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Article Abstract
Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.
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| http://dx.doi.org/10.1016/j.pep.2019.105543 | DOI Listing |
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Division of Cellular and Structural Biology, ICMR-National Institute for Research in Reproductive and Child Health, Parel, Mumbai, India.
Background: Mammalian cysteine-rich secretory proteins (CRISPs) are predominantly expressed in the male reproductive tract. Knockout mice lacking two or more CRISPs show defects in sperm transport, sperm-egg interaction and Ca homeostasis. CRISPs play redundant and specific roles via their binding partners.
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FASEB J
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Instituto de Biología y Medicina Experimental (IByME-CONICET), Ciudad Autónoma de Buenos Aires, Argentina.
Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2 .
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Protein Expr Purif
March 2020
The School of Biological Sciences, Monash University, Clayton, Victoria, 3800, Australia. Electronic address:
Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield.
View Article and Find Full Text PDFRelevance of CRISP proteins for epididymal physiology, fertilization, and fertility.
Andrology
September 2019
Instituto de Biología y Medicina Experimental (IByME-CONICET), Buenos Aires, Argentina.
Background: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process.
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Materials And Methods: We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team.
Impaired male fertility and abnormal epididymal epithelium differentiation in mice lacking CRISP1 and CRISP4.
Sci Rep
December 2018
Instituto de Biología y Medicina Experimental (IByME-CONICET), Buenos Aires, C1428ADN, Argentina.
Epididymal Cysteine Rich Secretory Proteins 1 and 4 (CRISP1 and CRISP4) associate with sperm during maturation and play different roles in fertilization. However, males lacking each of these molecules individually are fertile, suggesting compensatory mechanisms between these homologous proteins. Based on this, in the present work, we generated double CRISP1/CRISP4 knockout (DKO) mice and examined their reproductive phenotype.
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