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

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. En...

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Veröffentlicht in:Plant molecular biology 1998-10, Vol.38 (3), p.379-391
Hauptverfasser: Tibbot, B.K. (Wisconsin Univ., Madison, WI (USA). Dept. of Agronomy), Henson, C.A, Skadsen, R.W
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Sprache:eng
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Zusammenfassung: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.
ISSN:0167-4412
1573-5028
DOI:10.1023/A:1006006203372