Characterization of recombinant high pI Barley α-Glucosidase

Henrik Næsted, Birte Svensson

    Research output: Contribution to conferencePosterResearch

    Abstract

    α-glucosidase activity in barley seeds plays a crucial role in embryonic development. The concerted action of α-glucosidase, α-amylase, β-amylase, and limit dextrinase serve as the machinery responsible for the supply of glucose as energy source for the developing embryo in the germinating seed (MacGregor A.W.). Here we present the recent results of the expression and characterization of the recombinant full length barley high pI α-glucosidase in Pichia Pastoris. In order to facilitate in the range of mg protein yield, a clone representing an N-terminal hexa histidine tagged recombinant form of the enzyme was grown under high cell-density fermentation conditions. This approach enabled a successful protein expression profile under the highly sensitive methanol utilization phase of the fermentation procedure. The enzyme was purified using a four step purification strategy. Interestingly, the purified enzyme exhibits a higher molecular mass than expected from its primary sequence when applied on SDS-PAGE, indicating a possible post translational modification of the recombinant α-glucosidase. Preliminary enzyme kinetic analysis has demonstrated that the purified α-glucosidase is “fully” active when compared to the kinetic data of the native enzyme (Frandsen et al.). The presented data illustrates for the first time the successful production of enzymatically active full length recombinant high pI barley α-glucosidase (Tibbot et al. and Fransen et al.). Frandsen et al. 2000 Plant Physiology 123, 275-286. MacGregor A.W. 1987 CRC Crit. Rev. Biotechnol. 5, 117-128. Tibbot et al. 1998 Plant Molecular Biology 38, 379-391.
    Original languageEnglish
    Publication date2004
    Publication statusPublished - 2004
    EventThe Plant Poly-Saccharide WorkShop - York, UK
    Duration: 1 Jan 2004 → …

    Conference

    ConferenceThe Plant Poly-Saccharide WorkShop
    CityYork, UK
    Period01/01/2004 → …

    Cite this

    Næsted, H., & Svensson, B. (2004). Characterization of recombinant high pI Barley α-Glucosidase. Poster session presented at The Plant Poly-Saccharide WorkShop, York, UK, .