DNA sequence of Mirabilis antiviral protein (MAP), a ribosome-inactivating protein with an antiviral property, from Mirabilis jalapa L. and its expression in Escherichia coli

• Published in: The Journal of biological chemistry 1991-05, Vol.266 (13), p.8426-8430
• Main Authors: Kataoka, J. (Japan Tobacco Inc., Yokohama, Kanagawa, Japan), Habuka, N, Furuno, M, Miyano, M, Takanami, Y, Koiwai, A
• Format: Article
• Language: English
• Subjects: ADN; Amino Acid Sequence; ANTIVIRAL; Antiviral Agents; Base Sequence; Biological and medical sciences; Blotting, Western; CLONACION; CLONAGE; Cloning, Molecular; DNA; Enzyme-Linked Immunosorbent Assay; Escherichia coli - genetics; Escherichia coli - growth & development; Fundamental and applied biological sciences. Psychology; Gene expression; genes; Introns; MAP protein; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; N-Glycosyl Hydrolases; Nucleic Acid Conformation; NUCLEOTIDE; NUCLEOTIDOS; NYCTAGINACEAE; Plant Proteins - genetics; Plant Proteins - metabolism; PROPIEDADES ANTIMICROBIANAS; PROPRIETE ANTIMICROBIENNE; Protein Precursors - genetics; Protein Precursors - metabolism; Protein Sorting Signals - metabolism; PROTEINAS VEGETALES; PROTEINE VEGETALE; Repetitive Sequences, Nucleic Acid; Ribosome Inactivating Proteins, Type 1; Ribosomes - drug effects; roots; Transformation, Bacterial; VIRICIDAS
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)92992-3

We cloned a cDNA for Mirabilis antiviral protein (MAP), a ribosome-inactivating protein (RIP), which inhibits the mechanical transmission of plant virus and the in vitro protein synthesis of both prokaryotes and eukaryotes. The cDNA consisted of 1066 nucleotides and could encode 278 amino acids. The major part of the amino acid sequence (from Ala29 to Ser278) was identical with the sequence of native MAP as determined by protein sequencing. An NH2-terminal extrapeptide (28 amino acid residues) of MAP was comparable with the signal peptides of plant proteins accumulating in the vacuole. A stable hairpin structure was predicted in the 3'-noncoding region of the cDNA. Tandem repeated sequences were found downstream from the hairpin structure. They were composed of triple complete repeats of a heptanucleotide with preceding and following hexa-nucleotide repeats. The cDNA was expressed in Escherichia coli based on the T7 expression system. The product encoded by the cDNA was confirmed to be MAP precursor by Western blotting followed by immunological analysis. The growth of the transformants was inhibited by the expression of the gene. MAP precursor also seemed to inhibit the protein synthesis of E. coli just as native MAP has been observed to do