Purification and characterization of CRISP-3 from human seminal plasma and its real-time binding kinetics with PSP94.
Cysteine-rich secretory proteins (CRISPs) have been postulated to have a role in male reproduction and prostate pathophysiology. Of the mammalian CRISPs, CRISP-3 levels in particular have been shown to be upregulated in prostate cancer. Efforts have been made to obtain highly pure CRISP-3 for gaining structure-function information of this protein. However, well characterized and highly pure protein is not available yet. CRISPs from snake venom have been purified using prostate secretory protein of 94 amino acids (PSP94) has been reported earlier. In the present study, CRISP-3 was purified to homogeneity from human seminal plasma using human PSP94-immnobilized affinity column. The molecular mass of the purified protein was determined by SDS-PAGE followed by immunoblotting and found to be ∼26kDa and ∼28kDa. The purity was further verified using MALDI-TOF MS analysis, where two peaks at m/z 25509 and 27715 were obtained. The lower molecular weight peak corresponds to the calculated molecular mass of CRISP-3 (∼26kDa); whereas the higher molecular weight peak was confirmed to be the glycosylated form (∼28kDa) from the deglycosylation experiment. Binding of PSP94 in increasing concentrations to purified CRISP-3 immobilized chip was further validated using surface plasmon resonance. The kinetics data suggested that purified CRISP-3 binds specifically and with high affinity to PSP94. In conclusion, a homogeneous preparation of highly pure CRISP-3 protein is obtained from human seminal plasma.
Division of Structural Biology, India.
Jenifer H
JH
Anklesaria
Division of Structural Biology, India.
Rushika R
RR
Pandya
Division of Structural Biology, India.
Bhakti R
BR
Pathak
Division of Structural Biology, India; ICMR Biomedical Informatics Centre, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai, 400 012, India. Electronic address: smitamahale@hotmail.com.
Smita D
SD
Mahale
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008297:Male; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D029584:Prostatic Secretory Proteins / Q000737:chemistry / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012661:Semen / Q000737:chemistry; D029589:Seminal Plasma Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
C489926:CRISP3 protein, human; D029584:Prostatic Secretory Proteins; D012471:Salivary Proteins and Peptides; D029589:Seminal Plasma Proteins; C043076:beta-microseminoprotein; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Binding protein; CRISP-3; Human seminal plasma; PSP94; Prostate; Purification