Heterologous expression and biophysical characterization of soluble oligosaccharyl transferase subunits.
Oligosaccharyl transferase (OT) catalyzes the first committed step in N-linked protein glycosylation, a co-translational process that occurs in the lumen of the endoplasmic reticulum. The yeast Saccharomyces cerevisiae enzyme complex comprises nine integral membrane proteins, five of which are essential for catalysis. Due to the challenges with purifying the active enzyme complex for detailed biophysical studies, a systematic study to express, isolate, and characterize the soluble domains of three of the largest subunits in the complex (Nlt1p, Wbp1p, and Swp1p) is reported. The proteins are expressed using the lytic baculovirus expression system and the new constructs are well behaved, monomeric in solution, and glycosylated. Two of the proteins interact with each other as seen by gel filtration and circular dichroism. This study provides a framework to study the roles of these three essential subunits of the eukaryotic OT complex.
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Robert E
RE
Dempski
Barbara
B
Imperiali
D016367:Baculoviridae; D015153:Blotting, Western; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D008565:Membrane Proteins; D014415:Tunicamycin; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase