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Cloning and functional expression of Taiwan cobra chymotrypsin inhibitorCheng, Yun-Ching 20 June 2003 (has links)
Previous studies showed that dendrotoxins and B chain of b-Bungarotoxin shared sequence and structural homology with Kunitz-type protease inhibitors. In the present study, the cDNA of Kunitz¡Vtype protease inhibitor was successfully amplified from Taiwan cobra venom gland total RNAs using the primers designed from the B chain of b-Bungarotoxin. The deduced amino acid sequence of the cDNA exhibited the structural character of chymotrypsin inhibitor, and the mature protein contained 57 amino acids with six Cys residues. The chymotrypsin inhibitor was subcloned into pET29a(+) and transformed into BL21(DE3) E.coli strain. The expressed protein was isolated from inclusion bodies of E.coli and subjected to refolding into its folded structure. The inhibitor potency of the recombinant protein on chymotrypsin activity had a Ki value of 461.3 mM. However, removal of its N-terminal fused peptide with thrombin further increased the Ki value to 31.7 mM. Removal of the N-terminal residues further reduced its inhibitory potency, and the inhibitory activity completely lost after deleting three residues at the N-terminus of mature protein. This reflects that the N-terminal region of protease inhibitor should be associated with its activity. The genomic DNA encoding the precursor of the inhibitor was also amplified using PCR. The genetic structure composed of three exons and three introns, which shared the same organization with the b-Bungarotoxin B chain gene. Moreover, the two genes showed a high degree of sequence identity up to 83%. This observation emphasizes the idea that the B chain of b-Bungarotoxin and protease inhibitor are evolutionarily related.
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Structural and functional involvement of N-terminal region in the enzymatic activity of Taiwan cobra phospholipase A2Chiou, Yi-ling 10 August 2006 (has links)
The goal of the present study is to explore the functional involvement of the N-terminal region in the biological activity of phospholipase A2 (PLA2) enzyme. Native PLA2 from the venoms of Naja naja atra and Bungarus multicinctus and N-terminally mutated N. naja atra PLA2, i.e. an additional Met before Asn-1(M-PLA2), substitution of Asn-1 with Met-1(PLA2(N1M)) and removal of N-terminal seven residues (PLA2(¡µN7)), were employed in this study. Mutations on the N-terminal region insignificantly perturbed the binding ability of PLA2 for Ca2+ and ANS, but the enzymatic activity of mutants drastically decreased. Moreover, an alteration in the secondary structure was observed as revealed by CD spectra. Compared to other mutants, the fine structure of Ca2+-binding site within PLA2(¡µN7)) changed. Additionally, removal of the N-terminal region caused significant alternation in the structures of active site and substrate-binding site as evidenced by the results of fluorescence measurement, chemical modification and denaturation with detergents. In all N-terminal mutants, substituting Ans-1 with Met-1 affected the NNA-PLA2 structure to a least extent. The membrane-damage activity of PLA2(N1M) and M-PLA2 was 89% and 34% that of NNA-PLA2, respectively. PLA2(¡µN7) did not exhibit the membrane-damage activity. Studies on the biological activities of chemically modified N. naja atra PLA2 reflected a dissociation of the enzymatic activity from membrane-damage activity, and suggested the involvement of Trp-18, Trp-61, Lys-65, Tyr-3 and Tyr-63 in membrane-damage activity. Collectively, our data indicate that the intact N-terminus was crucial for maintaining of the functional conformation of PLA2 in the manifestation of the enzymatic activity and membrane-damage activity, and the enzymatic activity of PLA2 is in aid of but not exclusively essential for the membrane-damage effect.
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