£\-Bungarotoxin (£\-Bgt), a £\-neurotoxin from Taiwan banded krait (Bungarus multicinctus), consists of a single polypeptide chain of 74 amino acid residues cross-linking with five disulfide bonds. In order to explore the structural dependence of the disulfide bonds in the toxin molecule, the mutants with deleting one out of the five disulfide bonds were prepared by site-directed mutagenesis. The mutated and wild-type cDNAs were subcloned into the expression vectors pET14b as well as pET32a(+), and then transformed into Escherichia coli, BL-21(DE3). The recombinant proteins derived from pET14b expression system were isolated from inclusion bodies of E. coli, and refolding into their folded structure in vitro. Alternatively, the proteins derived from pET32a(+) system were expressed as a fusion protein and purified on a His-Bind resin column. Moreover, the recombinant proteins were further purified using a reverse phase column, and the homogeneity of recombinant proteins was determined by SDS-PAGE analyses. Noticeably, the refolding reaction was beneficially achieved by deleting the disulfide bond Cys29-Cys33. Although SDS-PAGE analyses showed that the recombinant proteins were homogeneous in molecular weight, several disulfide isomers appeared in each protein preparation as revealed by native gel analyses. These observations suggest that the formation of disulfide bonds and folding of£\-Bgt may pass through multiple pathways.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0516101-172117 |
| Date | 16 May 2001 |
| Creators | Cheng, Ching-Wen |
| Contributors | C. C. Yang, C. C. Chang, L. S. Chang |
| 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-0516101-172117 |
| Rights | unrestricted, Copyright information available at source archive |
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