An illustration of optimal selected glycosidase for N-glycoproteins deglycosylation and crystallization

Glycoproteins are protein molecules that contain the carbohydrate portion (glycans) covalently attached to poly-peptide side-chains, which are assembled in the endoplasmic reticulum (ER) and the Golgi by a controlled sequence of glycosyltransferase and glycosidase processing reactions. The presence of N-glycans controls proper folding of both secretory and membrane-bound proteins. In vitro recombinant glycoproteins are normally badly-glycosylated in the insect or mammalian cell lines, especially the secretory proteins. These N-glycans are usually complex, chemically and conformationally heterogeneous and frequently detrimental to the formation of well-ordered crystal lattices, obstructing the development of protein crystallography. Here we present pattern recognition receptor (PRR) of Arabidopsis FLS2 ectodomain after glycosidases treatment can still heterodimerize with its co-receptor BAK1 induced by flg22 through gel-filtration analysis. Appropriate deglycosylation strategies will not impact the characteristic and native conformation of FLS2 recombinant glycoprotein in vitro assay. The data reveals that Endoglycosidase F (Endo F) but not N-glycosidase F (PNGase F) can reduce the N-glycans of the residues of FLS2 ectodomain in native state, providing the probability of enhancing crystallization or raising diffraction resolution by means of different glycosidase combination and optimization. (C) 2018 Elsevier B.V. All rights reserved.