Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing
