Molecular Cloning and Expression in Escherichia coli of cDNA Encoding the Subunit of Sweet Potato ^-Amylase1

Tuberous roots of the sweet potato are unusually rich in)9-amylase, and the £-amylase polypeptides account for about 5 % of the total soluble protein of the organ. Unlike /9-amylases from other origins, the sweet potato ^-amylase is a tetramer of identical subunits, and it also bears starch phosphorylase-inhibitor activity. A cDNA for the subunit of sweet potato /9-amylase was obtained by immunological screening of an expression cDNA library constructed by the vector-primer and linker method using a plasmid vector containing toc-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 /J-amylase which was identical in size with the mature subunit, and the /9-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 /9-amylase. An amino acid sequence identical to the N-terminal amino acid sequence of the mature subunit appeared immediate-ly after the initiator methionine of the precursor, indicating that the subunit of £-amylase is synthesized as a mature form. Comparison of the amino acid sequences of subunits o