Isolation, Characterization And Gene Cloning Of Mcofp3 From Mytilus Coruscus

Marine mussels attach securely to solid surfaces in the presence of moisture through a holdfast known as the byssus formed by variant kinds of foot proteins secreted from its foot glander, which makes mytilus foot proteins (mfp) increasingly popular in marine adhesive protein researches. 6 different foot proteins have been identified to date from mussels and named mfp1~6, respectively. Mfp3, the most abundant mfp family, locates mainly in the plaques, together with a newly found mfp family, mfp6, and they both play key roles in underwater adhesion between the byssus and surrounding materials. Possessing important economic value, Hard-shell mussel Mytilus coruscus, with strong byssul and excellent adhesion, widely spreads all through the littoral extent of Zhejiang, China, while its foot proteins had rarely been studied.To understand the composition and the characterization of adhesive proteins from Mytilus coruscus several foot proteins were separated by C18 reverse-phase HPLC from the acetic acid-urea extracts of plaques and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry to determine the mass ranging from 3 to 6 kDa followed with partially sequencing by Edman degradation. Three of these proteins, containing abundant amount of Dopa, were determined to be variants of a family known as mfp3 (mytilus foot protein 3) and the other three proteins were presumed to be novel types of foot proteins, because no results were matched after online BLAST search. Quartz crystal microbalance analysis assay showed that some of these proteins exhibited strong absorption abilities on gold surface, which directly correlated with their adhesion force.For better understanding of the gene composition and diversity mechanism of mcofp3 and mcofp6 from M. coruscus, The total RNA was extracted by TRIZOL and the mRNA was purified via affinity chromatograph with oligo(dT) cellulose; utilizing gateway technology double-strand cDNA fragments were harvested, concentrated and ligated with pCMV sport6 vector. Subsequently PCR amplifications were executed with specific primers designed according to high conserved gene regions and the cDNA library of the foot glander cells as the template. Afterwards 14 full-length cDNA sequences of mfp3 variants and 8 of mfp6 variants were obtained followed by sequence alignment and variation sites analysis. The results showed that 14 mcofp3 genes were categorized as two subfamilies of mfp3 while the mcofp6 genes were highly conserved. This study build the foundation for further research on probing the adhesion and molecular diversity mechanism of the foot proteins from M. coruscus and the development of related new adhesive.