Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from pokeweed

A small cysteine-rich protein with antimicrobial activity was isolated from pokeweed (Phytolacca americana) seeds and purified to homogeneity. The pro tein inhibits the growth of several filamentous fungi and gram-positive bac teria. The protein was highly basic, with a pi higher than 10. The entire a mino acid sequence of the protein was determined to be homologous to antimi crobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. ameri cana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and sequenced. The deduced amino acid sequence shows the presence of a signal peptide at the amino terminus, suggesting that the protein is synthesized a s a preprotein and secreted outside the cells. The chromosomal gene shows t he presence of an intron located within the region encoding the signal pept ide. Southern hybridization showed that there was small gene family encodin g Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, an d was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted into the environment of the seeds during germination, and may create an in hibitory zone against soil-borne microorganisms. The disulfide bridges of P a-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicat es that the protein has a small cystine-knot folding, a positive patch, and a hydrophobic patch.