Purification, Characterization and Quantification of Yolk Proteins from Mature Ovaries of Mud Crabs (Scylla serrata)
K. Venkaiah
Department of Biotechnology, V.S. University, Nellore-524324, AP, India.
P. Gnana Prakasam
Department of Biotechnology, V.S. University, Nellore-524324, AP, India.
M. Hanuma Reddy
Department of Marine Biology, V.S. University, Nellore-524324, AP, India.
S.B. Sainath *
Department of Food Technology, V.S. University, Nellore-524324, AP, India and Department of Food Technology, V.S. University, Nellore-524324, AP, India.
*Author to whom correspondence should be addressed.
Abstract
The present study was aimed to purify, characterize and quantify vitellin from the mature ovaries of the mud crabs, Scylla serrata. Vitellin was purified using saturated ammonium sulfate method followed by dialysis. Analysis on SDS-PAGE revealed two subunits of 105 kDa and 85 kDa. To analyse the peptide profile of vitellin, the bands obtained were digested with trypsin and the tryptic peptides were sequenced by liquid chromatography-mass spectroscopy and MALDI-TOF analysis. The peptides identified from 105 kDa were spread along the entire sequence of vitellogenin corresponding to the full length of the protein. Purified vitellin was used to raise polyclonal antisera with which an enzyme linked immunosorbent assay method was developed. Typical standard curve using ELISA showed a detectable vitellin sensitivity range between 5.0 ng and 1.0 μg (linear regression analysis: R2= 0.991). Quantification of vitellogenin during natural reproductive cycle of mud crabs, revealed a significant increase (p< 0.001) in vitellogenin levels as compared to crabs at immature stage. To conclude, we demonstrated the isolation, purification, characterization and quantification of ovarian yolk protein from mud crabs, S. serrata which could be used as an aquaculture tool to identify and assess the signalling molecules that promote seed in hatchery industry.
Keywords: Aquaculture, crab, ELISA, ovaries, scylla serrata, vitellogenin