ABSTRACT
The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.
Subject(s)
Plasminogen Activators/genetics , Plasminogen Activators/metabolism , Polychaeta/enzymology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Western , Caseins/metabolism , Cloning, Molecular , DNA, Complementary/isolation & purification , Helminth Proteins/chemistry , Molecular Sequence Data , Open Reading Frames , Plasminogen Activators/chemistry , Polychaeta/genetics , Recombinant Fusion Proteins/isolation & purification , Sequence Alignment , Serine Endopeptidases/chemistry , Tylenchoidea/enzymologyABSTRACT
A novel big defensin was isolated and characterized from the plasma of Ruditapes philippinesis. It was purified to homogeneity by means of precipitation with (NH(4))(2)SO(4), gel-exclusion chromatography, two kinds of cation-exchange chromatography and named RPD-1. Its relative molecular mass was 24.8 kD by means of SDS-PAGE. By means of ABI437 amino acid sequence analyser, its 11 NH(2)-terminal amino acid sequence is AVPDVAFNAYG. Two databank systems (NCBI and EBI/EMBL) was indexed, no sequence with homology to RPD-1 was found. Furthermore, it exhibited strong inhibition on the growth of Gram-negative and -positive bacteria. Minimal inhibitory concentration (MIC) of RPD-1 against Staphylococcus aureus, Bacillus subtilis, Micrococcus tetragenus, Escherichia coli, Vibrio parahaemolyticus, Vibrio anguillarum was 9.6 mg/L, 76.8 mg/L, 38.4 mg/L, 76.8 mg/L, 19.2 mg/L, 19.2 mg/L respectively.
