Purification and characterization of a plant antimicrobial peptide expressed in Escherichia coli.
MiAMP1 is a low-molecular-weight, cysteine-rich, antimicrobial peptide isolated 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 arginine tRNA. The peptide was readily isolated in high yield from the insoluble fraction of the Escherichia coli extract. 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 fungi in vitro in a similar manner to the native peptide. To our knowledge, MiAMP1 is the first antimicrobial peptide from plants to be functionally expressed in E. coli. This will permit a detailed structure-function analysis of the peptide and studies of its mode of action on phytopathogens.
Cooperative Research Centre for Tropical Plant Pathology, University of Queensland, Brisbane, 4072, Australia.
S J
SJ
Harrison
A M
AM
McManus
J P
JP
Marcus
K C
KC
Goulter
J L
JL
Green
K J
KJ
Nielsen
D J
DJ
Craik
D J
DJ
Maclean
J M
JM
Manners
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli; D005658:Fungi / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008970:Molecular Weight; D009754:Nuts / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D014197:Trees / Q000235:genetics
C105617:AMP1 protein, Macadamia integrifolia; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins