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Open AccessArticle

The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity

Department of Biological Sciences, P.O. Box 70703, East Tennessee State University, Johnson City, TN 37614, USA
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Author to whom correspondence should be addressed.
Plants 2020, 9(3), 402; https://doi.org/10.3390/plants9030402
Received: 11 March 2020 / Revised: 21 March 2020 / Accepted: 22 March 2020 / Published: 24 March 2020
(This article belongs to the Special Issue Advances in Citrus Research)
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. View Full-Text
Keywords: glucosyltransferase; recombinant tags; His-tag; flavonoids; flavonol; grapefruit; C-terminal recombinant tags; pH optima; reaction kinetics glucosyltransferase; recombinant tags; His-tag; flavonoids; flavonol; grapefruit; C-terminal recombinant tags; pH optima; reaction kinetics
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MDPI and ACS Style

Birchfield, A.S.; McIntosh, C.A. The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity. Plants 2020, 9, 402. https://doi.org/10.3390/plants9030402

AMA Style

Birchfield AS, McIntosh CA. The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity. Plants. 2020; 9(3):402. https://doi.org/10.3390/plants9030402

Chicago/Turabian Style

Birchfield, Aaron S.; McIntosh, Cecilia A. 2020. "The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity" Plants 9, no. 3: 402. https://doi.org/10.3390/plants9030402

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