8S Globulin of Mungbean [Vigna radiata (L.) Wilczek]: Cloning and Characterization of Its cDNA Isoforms,Expression in Escherichia coli,Purification,and Crystallization of the Major Recombinant 8S Isoform

摘要

Three isoforms of the cDNA of the major 8S globulin of mungbean,8Sa,8Salpha',and 8Sbeta,were isolated,cloned,and characterized.The cDNA sequences of 8Sa,8Salpha',and 8Sbeta had open reading frames of 1362,1359 or 1362,and 1359 bp,respectively,which code for 454,453 or 454,and 453 amino acids corresponding to molecular weights of 51 973,51 627 or 51 758,and 51 779,respectively.Homology in terms of cDNA and amino acid sequences was 91 -92% between 8Sa and 8Salpha',87% between 8Salpha and 8Sbeta,and 86-88% between 8Salpha' and SSbeta.The signal peptide was found to be 1 -25,1 -24 or 25,and 1 -23 for 8Salpha,8Salpha',and QSbeta,respectively,using the signalP website (Nielsen,H.Engelbrecht,J.Brunak,S.von Heijne,G.Protein Eng.1997,10,1-6).The propeptide was determined to be IVHREN.A single site for glycosylation (N-X-S/T) was observed about 90 amino acids from the C terminus.Homology between mungbean 8S isoforms and other 7-8S proteins ranged from 45 to 68% within members of the legume family and 29 to 34% for crops of different species.The major isoform 8Sa was expressed in Escherichia coll and purified by successive ammonium sulfate fractionation,hydrophobic interaction,and Mono Q column chromatography.The recombinant 8Sa,but not the native form,was successfully crystallized producing rhombohedral crystals.