Molecular cloning and expression of the mannose/glucose specific lectin from Castanea crenata cotyledons.
cDNA clones encoding a mannose/glucose specific lectin, CCA, from Castanea crenata cotyledons have been isolated and sequenced. The cloned CCA cDNA had an open reading frame of 927 bp encoding 309 amino acid residues. Compared with the amino acid sequence determined for the protein chemically, it was clarified that CCA has no signal peptide and undergoes no proteolytic cleavage as do other mannose specific Jacalin-related lectins. The coding region of CCA was introduced into an expression vector, pET-22b(+), and then transferred into Escherichia coli BL21(DE3). Although recombinant CCA (rCCA) accumulated as inclusion bodies, refolded rCCA exhibited a similar CD spectrum to nCCA and regained the hemagglutination activity. In addition, a hapten inhibition assay revealed that nCCA and rCCA showed the same specificities toward sugars and glycoproteins. On measurement by GPC-MALLS in the native state, the absolute molecular mass of nCCA was found to be 332 7 kDa, which indicated that nCCA is a decamer of identical subunits having a molecular mass of 33 kDa. The same as the natural molecule, rCCA showed a molecular mass of 320 +/- 5 kDa and was judged to also be a decamer. These results indicate that the rCCA obtained in this study is equivalent to nCCA.
Nakamura
Sachiko
S
Graduate School of Science and Technology, Faculty of Agriculture, Kobe University, Rokkodai-chou 1, Nada-ku, Kobe 657-8501, Japan.
Ikegami
Ayako
A
Matsumura
Yasuki
Y
Nakanishi
Tetsu
T
Nomura
Keiichi
K
D000372:Agglutination Tests; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018548:Cotyledon / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry*; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D037102:Lectins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D037121:Plant Lectins; D010946:Plants, Medicinal / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D037102:Lectins; D037121:Plant Lectins; D011994:Recombinant Proteins; D005947:Glucose; D008358:Mannose