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Updated: May 12, 2025
High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Published on: December 28, 2015
Expression in HEK293 cells, purification, and characterization of recombinant human α-glucosidases
So Nishimoto1, Teisuke Takita1, Kazumichi M Nishida2
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
Abstract:
In mammals, intestinal α-glucosidase exists within maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) complexes. Here, we purified recombinant human MGAM and SI from human embryonic kidney 293 cells using Flag tag-based affinity chromatography. Steady-state kinetic analysis at pH 6.0 and 37 °C showed that the maltase activities of MGAM and SI were higher than their sucrase and isomaltase activities.
Insights
Mammalian intestinal enzymes maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) were purified. Kinetic analysis revealed MGAM and SI exhibit higher maltase activity than sucrase or isomaltase activity.
Area of Science:
- Biochemistry
- Enzymology
- Mammalian physiology
Background:
- Intestinal alpha-glucosidases are crucial for carbohydrate digestion in mammals.
- These enzymes function as part of larger protein complexes, specifically maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI).
Purpose of the Study:
- To purify recombinant human MGAM and SI complexes.
- To characterize the enzymatic activities of purified MGAM and SI.
Main Methods:
- Recombinant human MGAM and SI were expressed in human embryonic kidney 293 (HEK293) cells.
- Protein purification was achieved using Flag tag-based affinity chromatography.
- Steady-state kinetic analyses were performed at pH 6.0 and 37°C.
Main Results:
- Purified recombinant human MGAM and SI were successfully obtained.
- Kinetic analysis demonstrated that the maltase activities of both MGAM and SI were significantly higher than their respective sucrase and isomaltase activities.
Conclusions:
- The study successfully purified and characterized key human intestinal disaccharidases.
- MGAM and SI display a preferential catalytic activity towards maltose compared to sucrose and isomaltose.
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