Properties of a ribosome-inactivating protein, gelonin, purified using three different methods.
Ribosome-inactivating protein, gelonin, isolated from an Indian plant Gelonium multiflorum of Euphorbiaceae family has been used to design and synthesize immunotoxins and hormonotoxins for selective targeting purposes. Since gelonin isolated by aqueous extraction, cation-exchange chromatography and gel-filtration chromatography (Method I), contains non-proteinous material absorbing at 280 nm, the ammonium sulphate precipitation method (Method II) and Cibacron blue affinity chromatography method. (Method III) have been used to purify gelonin from the dry seeds. Three batches of gelonin purified by each method were prepared and subjected to extensive physico-chemical and immunochemical characterization. The molecular weight was determined by gel-filtration chromatography on a pre-calibrated Sephadex G-100, TSK-G4000 TW on HPLC or Superose-12 on fast protein liquid chromatography. In all cases, the molecular weight was approximately 30,000Da. The SDS-PAGE also revealed a homogeneous protein of 30kDa molecular weight. In Method II, the non-proteinous material which binds to CMC-gel in association of gelonin was substantially removed during ammonium sulphate fractionation. A careful analysis clearly revealed that Method II, although yielded low protein, gave gelonin devoid of the non-proteinous material. The SPDP modification of epsilon-NH2 groups of gelonin obtained from Methods I, II, and III was also carried out and its effect on immunoreactivity was studied.
Singh
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Institute of Self-Organising Systems and Biophysics, North-Eastern Hill University, Shillong, Meghalaya, India.
Kar
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D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000737:chemistry*; D014118:Toxins, Biological
D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological; C051829:bryodin; C025674:GEL protein, Gelonium multiflorum