Isolation, Purification and Crystallization of Ribosome inactivating protein from Barley (Hordeum vulgare)

Department of Crystallography and Biophysics, University of Madras
â—‹Etti Sundaresan Karthe Ponnuraj Shanmugam Guruswamy

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in plants, fungi, and bacteria. RIPs inhibit protein synthesis by virtue of their enzymatic activity, selectively cleaving a specific adenine residue from a highly conserved, surface-exposed, stem-loop (S/R loop) structure in the 28S rRNA of ribosomes. RIP's are usually classified into two groups: type I and type II. Type I (or single-chain) RIP's are unable to enter intact cells and thus are only capable of intoxicating cell-free systems. Type I RIP's are thought to be better candidates than type II RIP's for constructing immunotoxins because they lack their own cell recognition and binding ability. RIP from barley seeds, belonging to the type I category, inactivates eukaryotic ribosomes via a mechanism identical to that of ricin A-chain. Like many other type I RIP's, it is a basic protein with pl of above 9.0 The mature polypeptide chain, consisting of 280 amino-acid residues, has a molecular mass of about 30 kDa. The isolation and purification of RIP from barley seeds was done by a simple procedure of Ammonium sulphate precipitation and Ion exchange column chromatography using CM-Sephadex as the column material and the single peak of the protein was confirmed by 15% SDS-PAGE gel electrophoresis. The micro crystals of RIP obtained by hanging drop vapor-diffusion method using PEG 4000 as the precipitant in reservoir. Improving the quality of crystals is in progress.