Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue

• Published in: Plant molecular biology Vol. 38; no. 3; pp. 379 - 391
• Main Authors: Tibbot, B.K. (Wisconsin Univ., Madison, WI (USA). Dept. of Agronomy), Henson, C.A, Skadsen, R.W
• Format: Journal Article
• Language: English
• Published: Netherlands Kluwer Academic Publishers 01-10-1998
• Subjects: ADN RECOMBINADO; ADN RECOMBINE; ALFA GLUCOSIDASA; ALPHA GLUCOSIDASE; alpha-Glucosidases - genetics; alpha-Glucosidases - immunology; alpha-Glucosidases - metabolism; Amino Acid Sequence; ANTICORPS POLYCLONAL; ANTICUERPOS POLICLONALES; barley; Base Sequence; complementary DNA; DNA, Complementary - genetics; DNA, Plant - genetics; ESCHERICHIA COLI; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; GENE EXPRESSION; genes; GERMINACION; GERMINATION; gibberellic acid; Hordeum - enzymology; Hordeum - genetics; HORDEUM VULGARE; Immunochemistry; isomaltose; MALTOSA; MALTOSE; methanol; Molecular Sequence Data; Molecular Weight; PICHIA; Pichia - genetics; Pichia pastoris; POLYCLONAL ANTIBODIES; proteins; RECOMBINANT DNA; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; SACCHAROMYCES CEREVISIAE; seed germination; Seeds - enzymology; trehalose; yeasts
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/A:1006006203372

An α-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 α-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant α-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 α-glucosidase. The enzyme had a Kₘ of 1.88 mM and Vₘₐₓ of 0.054 µmol/min on maltose. The recombinant α-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley α-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa α-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 α-glucosidase enzyme activity.Abbreviations: AGL, barley seed α-glucosidase; rAGL, recombinant barley seed α-glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.