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.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0620103-142902 |
| Date | 20 June 2003 |
| Creators | Cheng, Yun-Ching |
| Contributors | Chun-Chang Chang, Leng-Sen Chang, Wen-Chun Hung |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0620103-142902 |
| Rights | withheld, Copyright information available at source archive |
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