Rice bifunctional alpha-amylase/subtilisin inhibitor: cloning and characterization of the recombinant inhibitor expressed in Escherichia coli.
The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.
Yamasaki
Teruyuki
T
Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Japan. yamagata@kobe-u.ac.jp
Deguchi
Masaki
M
Fujimoto
Toshiko
T
Masumura
Takehiro
T
Uno
Tomohide
T
Kanamaru
Kengo
K
Yamagata
Hiroshi
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D020860:Subtilisin / Q000037:antagonists & inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C076389:subtilisin inhibitor p20, Oryza sativa; D000516:alpha-Amylases; D020860:Subtilisin