The N-terminal fragments carrying N40D variants were expressed by using an expression vector for extracellular secretion in mammalian cells. Briefly, the soluble N-terminal region of MOR N40 and D40 fused with a C-terminal IgG Fc domain to permit affinity chromatography was inserted in expression vector p-cDNA 3.1. This construct has been further modified to introduce a 3Cpro cleavage site upstream from the Fc domain as described previously 30. Both N-terminal MOR constructs (N40 and D40) contained the original expression vector, an N-terminal FLAG tag, signal peptide sequence and Fc sequence. They were expressed in HEK293 and HEK293 GnT1- (lacking the N-acetylglucosaminyltransferase I gene needed for high-order N-linked glycosylation processing) cells.
