| Autor: |
Harrison, Stuart J., McManus, Ailsa M., Marcus, John P., Goulter, Ken C., Green, Jodie L., Nielsen, Katherine J., Craik, David J., Maclean, Donald J., Manners, John M. |
| Zdroj: |
Protein Expression and Purification; March 1999, Vol. 15 Issue: 2 p171-177, 7p |
| Abstrakt: |
MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated from the nut kernel ofMacadamia integrifolia.A DNA sequence encodingMiAMP1 with an additional ATG start codon was cloned into a modified pET vector under the control of the T7 RNA polymerase promoter. The pET vector was cotransformed together with the vector pSB161, which expresses a rare arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of theEscherichia coliextract. The purified peptide was shown to have an identical molecular weight to the native peptide by mass spectroscopy indicating that the N-terminal methionine had been cleaved. Analysis by NMR spectroscopy indicated that the refolded recombinant peptide had a similar overall three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phytopathogenic fungiin vitroin a similar manner to the native peptide. To our knowledge,MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed inE. coli.This will permit a detailed structure–function analysis of the peptide and studies of its mode of action on phytopathogens. |
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