Exploring The Structures, Coding Genes And Characteristics Of Lumbricus Bimastus Nucleases EWD_1,2,3

Through previous research on Lumbricus Bimastus, 5 nuclease like enzymes were purified and named respectively EWD1,2,3 and EWN1,2. The enzymic kinetics properties, affective factors, substrate specificities of these enzymes have been explored. Based on the sources, molecular weights, pI (isoelectric point), characters of chromatography and electrophoresis, substrate specificities of these enzymes, it can be concluded that many kinds of nuclease-like enzymes have been found in the tissues of Lumbricus Bimastus.Objective:In order to validate the natures and structures of these proteins and obtain their coding genes, techniques of chromatography purification, 1D-PAGE, 1D-SDS-PAGE, 2D-PAGE, 2D-SDS-PAGE, proteinic chemistry, MALDI-TOF-MS, LC-MS/MS, cDNA library and RT-PCR were used on the studies of five aspects of EWD1,2,3.Methods and Results:1. Preparation of Lumbricus Bimastus nucleases with high purityA great quantity of enzyme samples with high purity (above 99.5%) are needed to explore the characters of proteins. Through cation exchange HPLC, 1-D-PAGE, 2-D-PAGE, non-denatured imidazole-sulfate-zinc reverse stain and recovery from gels, EWD1,2,3 with purity above 99.5% degree were prepared. Practicable methods of high-purity purification were established. The yeild rates of the three proteins were 27.9% (EWD1), 16.7% (EWD2) and 16.7% (EWD3) respectively; purification folds were 11.6 (EWD1), 11.2 (EWD2), 18.6 (EWD3) respectively, these provided enough samples for further research.2. Study on the characters of Lumbricus Bimastus nucleases EWDsMALDI-TOF-MS was used to determine the purities of the three proteins, the results were adequate for molecular weights and peptide mass fingerprintings (PMFs) examinations. The results showed that the accurate MWs of the three nucleases were 157191.388,69074.741 and 63461.35 respectively. The PMFs of EWD1 and EWD2 were obtained through protein enzymolysis, no matched proteins was found after Sequest database searching, this indicated that there is no information of the two proteins in the present protein database, thus, the two proteins were speculated as novel proteins. Characters of the three enzymes were compared with present...