Molecular cloning and expression in Escherichia coli of cDNA encoding the subunit of sweet potato beta-amylase.
Tuberous roots of the sweet potato are unusually rich in beta-amylase, and the beta-amylase polypeptides account for about 5% of the total soluble protein of the organ. Unlike beta-amylases from other origins, the sweet potato beta-amylase is a tetramer of identical subunits, and it also bears starch phosphorylase-inhibitor activity. A cDNA for the subunit of sweet potato beta-amylase was obtained by immunological screening of an expression cDNA library constructed by the vector-primer and linker method using a plasmid vector containing tac-SP6 promoters. The SP6 transcript of a 2,000 base-pair-long cDNA insert directed the synthesis in vitro of a precursor to the subunit of beta-amylase which was identical in size with the mature subunit, and the beta-amylase mRNA detected by Northern blot hybridization was identical in size with the SP6 transcript of the cDNA insert. The cDNA insert contained 1,494 base pairs of an open reading frame which codes for the 499-amino-acid-long precursor to the subunit of beta-amylase. An amino acid sequence identical to the N-terminal amino acid sequence of the mature subunit appeared immediately after the initiator methionine of the precursor, indicating that the subunit of beta-amylase is synthesized as a mature form. Comparison of the amino acid sequences of subunits of sweet potato beta-amylase and seed beta-amylases from barley and soybean indicated that these enzymes share about 68% amino acid identities among each other. Escherichia coli cells harboring the cDNA clone produced the mature-sized subunit of the beta-amylase, and the soluble extract exhibited amylolytic activity which migrated to the same position as the beta-amylase purified from the sweet potato in non-denaturing polyacrylamide gel containing soluble starch indicating that oligomerization of the subunit occurred properly in E. coli cells.
Yoshida
N
N
Laboratory of Biochemistry, School of Agriculture, Nagoya University, Aichi.
Nakamura
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D016415:Sequence Alignment; D014675:Vegetables / Q000201:enzymology*; D001614:beta-Amylase / Q000235:genetics*
D012333:RNA, Messenger; D004247:DNA; D001614:beta-Amylase