Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli

MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isol ated from the nut kernel of Macadamia integrifolia. A DNA sequence encoding MiAMP1 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 a rginine tRNA. The peptide was readily isolated in high yield from the insol uble fraction of the Escherichia coil extract. The purified peptide was sho wn to have an identical molecular weight to the native peptide by mass spec troscopy indicating that the N-terminal methionine had been cleaved. Analys is 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 fungi in vitro in a s imilar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coil. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens. (C) 1999 Academic Press.