Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue.
An alpha-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae alpha-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant alpha-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5-6.3 with an optimum at pH 4.3, classifying the enzyme as an acid alpha-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 micromol/min on maltose. The recombinant alpha-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley alpha-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa alpha-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in alpha-glucosidase enzyme activity.
Tibbot
B K
BK
Department of Agronomy, University of Wisconsin, Madison 53706, USA.
Henson
C A
CA
Skadsen
R W
RW
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics*; D007120:Immunochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D000520:alpha-Glucosidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D018076:DNA, Complementary; D018744:DNA, Plant; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000520:alpha-Glucosidases