Expression, purification, and enzymatic characterization of human UDP-glucose:glycoprotein glucosyltransferase-selenoprotein F complex from Sf9 insect cells

Abstract

Appropriate functioning of the glycoprotein quality control system in the endoplasmic reticulum is crucial for maintaining cellular homeostasis. UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) is a key component of this system, serving as a “folding sensor" by recognizing the partially folded state of cognate glycoproteins and reglucosylating their deglucosylated N-glycans. Notably, UGGT1 is known to form heterodimers with Selenoprotein F (SelenoF), although the mechanism underlying the complex formation remains unclear. To date, there have been no reports of large-scale expression systems for recombinant human UGGT1, and the formation of the human UGGT1-SelenoF complex has not been demonstrated in vitro. To address this, we used an insect cell-based expression system for heterologous expression of human UGGT1 and SelenoF, achieving high purity and efficiency superior to that of Escherichia coli expression systems. Importantly, the two proteins, prepared independently, were observed to form complexes in vitro. The establishment of a system for large-scale production of human UGGT1 and UGGT1-SelenoF complexes paves the way for future studies investigating their structural characteristics and dynamic behavior.