Prokaryotic Expression And Charac- Terization Of A Novel Cystatin In The PROG, Ceratophrys Cranwelli

Amphibians are natural resources for being sustainable utilization that have important value in economy, science, ecology and civilization. Amphibian resources are rich on the earth,and they are not only the important part of biological resources in the world, but also precious for human beings. Amphibian skins are pharmacologically active organs display-ing multiple defensive functions reasonable and essential for survival. Their skin glands have abundant secretions containing bioactive molecules with a large amount of structural and functional diversity.Several families of amphibian skin peptides, including bombesins, protease inhibitors, antimicrobial peptides, tachykinins, cholecystokinin, bradykinins, an-tioxidant peptides and gene-encoded neurotoxin, have been identified in recent years, but few cystatins have been extracted.This research target Ceratophrys cranwelli, a common ceratophrys in South America. We constructed its skin cDNA library. Under the premise of a known amino acid sequence named cystatin-cp screened from C. cranwelli skin cDNA library (The correspond peptide primary structure to the AAS has 41% homology with cystatin B that come from Nereis succinea by amino acid sequence comparison in NCBI), we designed the primer,cloned this amino acid sequence and constructed its prokaryotic expression system successfully.By pET-32a expression system of E.coli., cystatin-cp added with His-tag was induced well. After released from the fusion by formic acid cleavage, impure cystatin-cp had been acquired. Subsequently, we got purified cystatin-cp with cystatin activity through Sephadex G-50 and RP-HPLC. Mass spectrometry analysis was performed to identify the recombi-nant cystatin-cp and the molecular weight is not consistent with the prediction. With a scrupulous analysis of the mature peptide amino acid sequence, we have a guess that the site cleaved by formic acid is between 8Gly and 9Pro but not DP site in the pepetide, and it was added with a metal ion when ionized by MS. The Ki value for cystatin-cp is about 27.594μM, which is determined by Dixon plot.