Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds.
A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.
Sawano
Yoriko
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Miyakawa
Takuya
T
Yamazaki
Hiroshi
H
Tanokura
Masaru
M
Hatano
Ken-ichi
K
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification*; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004220:Disulfides / Q000737:chemistry; D020441:Ginkgo biloba / Q000737:chemistry*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; D004220:Disulfides; D011480:Protease Inhibitors; C519081:ginkbilobin-2 protein, Ginkgo biloba