IMPROVED CRYSTALS OF THE TOXIC PROTEIN MAP BY PROTECTION ENGINEERING TOWARDS THE HOST-SPECIFICITY

Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F whi ch exhibits a lower (1/100) activity against E. coli ribosomes while r etaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F M AP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the nati ve MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50 % ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)2 1) with a = b = 104.1 and c = 134.3 angstrom. The crystals are isomorp hous with the wild-type crystals but diffract to higher resolution. Im aging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.