Expression of functional recombinant scorpion β-neurotoxin Css II in E. coli
| Autor: | Ted T. Sakai, Tonny M. Johnson, N. Rama Krishna, Michael W. Quick |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Circular dichroism
Physiology Molecular Sequence Data Neurotoxins Scorpion Venoms Reptilian Proteins Biology medicine.disease_cause Binding Competitive Biochemistry Inclusion bodies law.invention Cellular and Molecular Neuroscience Endocrinology law Gene expression Escherichia coli Genes Synthetic medicine Amino Acid Sequence Nuclear Magnetic Resonance Biomolecular Expression vector Base Sequence Toxin Molecular biology Recombinant Proteins Solubility Recombinant DNA Biological Assay Thioredoxin |
| Zdroj: | Peptides. 21:767-772 |
| ISSN: | 0196-9781 |
| DOI: | 10.1016/s0196-9781(00)00206-0 |
| Popis: | The gene for a β-neurotoxin [ Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [ 3 H]GABA [γ-aminobutyric acid (GABA)] in neuronal cells as effectively as natural β-toxins. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect