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

• Published in: Journal of agricultural and food chemistry 2004-05, Vol.52 (9), p.2552-2560
• Main Authors: Bernardo, Amy Emiliana N, Garcia, Roberta N, Adachi, Motoyasu, Angeles, Jorge Gil C, Kaga, Akito, Ishimoto, Masao, Utsumi, Shigeru, Tecson-Mendoza, Evelyn Mae
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Base Sequence; biochemical polymorphism; Biological and medical sciences; Cloning, Molecular; complementary DNA; Crystallization; DNA, Complementary - genetics; DNA, Plant - genetics; Escherichia coli; Escherichia coli - genetics; Fabaceae - chemistry; Fabaceae - genetics; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Gene Expression; gene transfer; globulins; molecular cloning; Molecular Sequence Data; mung beans; nucleotide sequences; Phylogeny; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Protein Isoforms - genetics; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; seeds; Sequence Homology; signal peptide; Vigna radiata
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf0305938

Three isoforms of the cDNA of the major 8S globulin of mungbean, 8Sα, 8Sα‘, and 8Sβ, were isolated, cloned, and characterized. The cDNA sequences of 8Sα, 8Sα‘, and 8Sβ 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 8Sα and 8Sα‘, 87% between 8Sα and 8Sβ, and 86−88% between 8Sα‘ and 8Sβ. The signal peptide was found to be 1−25, 1−24 or 25, and 1−23 for 8Sα, 8Sα‘, and 8Sβ, 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 8Sα was expressed in Escherichia coli and purified by successive ammonium sulfate fractionation, hydrophobic interaction, and Mono Q column chromatography. The recombinant 8Sα, but not the native form, was successfully crystallized producing rhombohedral crystals. Keywords: 8S globulin; cDNA; isoforms; vicilin; amino acid sequence; crystallization; purification; homology analysis; signal peptide; propeptide; mungbean; Vigna radiata L.; legumes