Identification And Cloning Of Three Bioactive Polypeptides From Skin And Eggs Of Batrachuperus Pachunii And Rana Grahami

Amphibian is a biological treasure with great potentials as well as important natural resources with ecological, research, economic and cultural value. A variety of bio-active substances from amphibian skins which play an important role in defense against invading factors have attracted significant attention of the researchers all over the world. In this study, three bioactive proteins/ploypeptides were identified by biochemical and molecular biological methods.Two antimicrobial peptides manifesting a broad spectrum of antimicrobial activity against various microorganisms have been isolated from skin secretions of Rana grahami. These antimicrobial peptides were named grahamin 1 and grahamin 2. Their primary structures are GLLSGILGAGKNIVCGLSGLC (1942Da) and GLLSGILGAGKHIVCGL SGLC (1965Da), respectively, determined by Edman degradation and mass spectrometry. They are structurally related to nigrocins identified from skin secretions of the darkspotted frog, Rana nigromaculata. The cDNA clones encoding the precursor of grahamins were screened and sequenced from the skin cDNA library of R. grahami. The amino sequences deduced from the cDNA sequences match well with the results from Edman degradation. As other antimicrobial peptides from Rana species, grahamins contain a C-terminal loop region delineated by an intra-disulfide bridge named Rana box. Based on structural comparison of grahamin with other known antimicrobial peptides, grahamins could be classified into the family of antimicrobial peptides containing a single intra-disulfide bridge.Two proteins with an apparent molecular weight of 40kDa and 14.4kDa were purified from the egg of Rana grahami by a combination of gel filtration chromatography, AKTA(?) Resource-Q ion chromatography and reverse phase high performance liquid chromatography (RP-HPLC) steps. The purified two proteins showed a trypsin inhibitor...