Purification and Characterization of a Plant Antimicrobial Peptide Expressed in Escherichia coli Stuart J. Harrison,* Ailsa M. McManus,† John P. Marcus,* Ken C. Goulter,* Jodie L. Green,* Katherine J. Nielsen,† David J. Craik,† Donald J. Maclean,* and John M. Manners* ,1 *Cooperative Research Centre for Tropical Plant Pathology and †Centre for Drug Design and Development, University of Queensland, Brisbane 4072, Australia Received June 2, 1998, and in revised form September 28, 1998 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 over- all three-dimensional structure to that of the native peptide. The peptide inhibited the growth of phyto- pathogenic 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 struc- ture–function analysis of the peptide and studies of its mode of action on phytopathogens. © 1999 Academic Press Cysteine-rich, low-molecular-weight, antimicrobial peptides have been isolated from several plant, animal, and microbial sources. Many of these have been shown to possess inhibitory activity toward phytopathogens and expression of these peptides has the potential to confer disease resistance in transgenic plants (1,2). Antimicrobial peptides are either produced constitu- tively in some plant organs, e.g., seeds (1), or synthe- sized locally and systemically after pathogen challenge (3). As such, antimicrobial proteins are most probably important components of the plant defense system. To fully understand the role of antimicrobial peptides in plant defense it is important to develop techniques for the study of their mode of action and function. The peptide MiAMP1 has been isolated from the nut ker- nels of Macadamia integrifolia and has in vitro inhib- itory activity against numerous commercially impor- tant plant pathogens (4). In this report we describe a method for the production of this peptide in vitro. Large quantities of proteins and peptides are often necessary to allow full characterization of biological activity, three-dimensional structure, and mode of ac- tion. Until recently, the only way to obtain large quan- tities of MiAMP1 was to extract it from nut kernels. This is laborious and yields little protein. Recombinant DNA techniques permit the production of peptides and proteins in microorganisms so that large quantities can be purified. There are some reports of the production of antimicrobial peptides from animals in Escherichia coli (5) and yeast (6) but there is only one report of the in vitro expression and production of a plant antimi- crobial peptide (7). In this case, a plant defensin orig- inally purified from radish seeds was expressed in yeast. The radish defensin was expressed in yeast from a gene that encoded a preproprotein precursor from which the mature peptide was produced and secreted (7). E. coli provides a potentially simpler system for the expression of plant antimicrobial peptides and may be of more general use than yeast for the expression of peptides with inhibitory activity against fungi. How- ever, to our knowledge the expression of functional antimicrobial peptides of plant origin in E. coli has not been reported. Previously we have shown that the plant antimicrobial peptide MiAMP1 inhibited the growth of baker’s yeast but was inactive against most gram-negative bacteria including E. coli (4). Herein, we 1 To whom correspondence should be addressed at Cooperative Research Centre for Tropical Plant Pathology, Level 5, John Hines Building, University of Queensland, Brisbane 4072, Australia. Fax: 61-7-33654771. E-mail: j.manners@tpp.uq.edu.au. Protein Expression and Purification 15, 171–177 (1999) Article ID prep.1998.0992, available online at http://www.idealibrary.com on 171 1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.