Abstract
Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5–9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima.
Highlights
Plants produce secondary metabolites that impart a wide range of beneficial effects and increase overall survivability
Results testing recombinant Citrus paradisi flavonol-specific 3-O glucosyltransferase (Cp3GT) expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima
Cp3GT expressed in E. coli and the enzyme expressed in the Pichia system showed different pH optima
Summary
Plants produce secondary metabolites that impart a wide range of beneficial effects and increase overall survivability. Initial characterizations of pH optima for Cp3GT, obtained through expression in E. coli, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH. This agrees with other flavonoid glucosyltransferase (GT) pH optima of 6.5–8 [25,26,27]. More recent characterizations of Cp3GT expressed using P. pastoris, showed a higher optimal pH of 8.5–9 This suggests either an effect due to the location and chemistry of the recombinant tags, or an effect related to physiological differences between the cell expression systems [24]. This research was designed to test the hypothesis that recombinant tags have an effect on Cp3GT activity with respect to pH optima, substrate preference, kinetic parameters, and interaction with various metals
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