Purification of an osmotin-like protein from the seeds of Benincasa hispida and cloning of the gene encoding this protein

A pathogenesis-related (PR) protein was purified from the seeds of Benincas a hispida, which is a medicinal plant and a member of the Cucurbitaceae fam ily. Purification was achieved by using a procedure consisting of an acid t reatment step followed by two chromatography steps. The protein is a basic protein with molecular mass of similar to 28 kDa. The sequences of the N-te rminal 30 amino acids and four peptides generated from protease digestion w ere determined. These sequences indicated that the protein is an osmotin-li ke protein (OLP). Osmotin and OLPs are members of the thaumatin-like, PR-5 family of the PR proteins. A genomic clone of the gene encoding the protein was isolated and sequenced. The predicted protein has a signal peptide of 18 amino acids, and the mature protein has a molecular mass of 24.8 kDa wit h an isoelectric point of 7.67. The protein has 17 cysteine residues, of wh ich 16 appear in the same positions as those appear in the sweet-tasting pr otein thaumatin and several other thaumatin-like proteins. Southern hybridi zation analysis indicated that the gene encoding the protein is a single co py gene. A computer-generated, three-dimensional model of the protein is pr esented. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.