The development of a purification procedure for Peptide:n-Glycosidase A from Prunus amygdalus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

Peptide-N4-acetyl-β-glucosaminyl) asparagine amidases cleave the amide bond between N-linked glycans at N-acetylglucosamine and asparagine, liberating intact oligosaccharide chains from glycoproteins. Although PNGase A is commonly used by glycobiologists for removal of N-Iinked glycans from plant sources, much less is known about it than about PNGase F, an enzyme that is more commonly used for deglycosylating proteins. New studies on PNGase A have been initiated, with the aim of carrying out complete biochemical and structural studies in order to determine the substrate specificity, isoelectric point, primary, secondary and tertiary structures. Comparisons will then be made with PNGase F, whose three-dimensional structure is known. The first step in these studies is therefore to obtain some pure protein and amino acid sequence. Although purification protocols have been published previously, it was difficult to produce a homogeneous preparation following these methods and they have hence been modified. The methods used are described in Chapter 2 and the results of four preparations, using almond meal and almond emulsin as starling materials, are reported in Chapters 3-6. Although PNGase A had not been purified to homogeneity, an active band excised from a native gel and analysed by SDS-PAGE showed four major bands. Which band represents PNGase A remains to be determined.