Expression, purification and characterisation of recombinant peptide:N-glycosidase F. : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Biochemistry at Massey University

Abstract

PNGase F (Peptide-N4-(N-acetyl-D-glucosaminyl) asparagine amidase F) is an amidohydrolase isolated from the extracellular medium of the Gram-negative bacterium Flavobacterium meningosepticum. The 34.8-kDa enzyme catalyses the complete and intact cleavage of asparagine-linked oligosaccharide chains from their associated proteins. A T7 promoter-based E. coli expression system was developed in which PNGase F was expressed as a fusion protein with a leader sequence from the ompA gene. The hexa-histidine-tagged PNGase F was correctly processed and exported to the E. coli periplasm and had a calculated molecular weight of 36.2 kDa. A single step purification using immobilised metal affinity chromatography yielded 8 mg of pure protein per litre of culture. The sequence of the PNGase F coding region from the CDC strain 3352 of F. meningosepticum was found to differ from a published sequence from another strain of the bacterium (ATCC 33958) in 57 positions. These differences between the two strains result in eight amino acid substitutions, which are mostly conservative in nature and are on the surface of the protein. Moreover, three potential N-glycosylation sites not present in the ATCC strain 33958 were detected in CDC strain 3352. The recombinant enzyme has similar characteristics of the native enzyme with a pH optimum of 8.5 and is strongly inhibited by Ag+, Cu2+, Fe3+ ions but not by sulfhydryl-targeting agents such as DTT and NEM. This indicates inhibition by these ions is probably through interactions with a histidine residue at position 193 that may be involved in substrate recognition or catalysis. The specific activity of the native PNGase F is about four times that of the recombinant protein which may be contributed to inhibition by components of the CompleteTM protease inhibitor tablets used in the enzyme preparation or due to modifications for cloning and purification. Using a discontinuous assay and a non-labelled 11-mer ovalbumin-derived glycopeptide as substrate, a rough estimate of the Michaelis constant (Km) for the recombinant PNGase F was determined to be 2.1 μM. An intriguing observation with the activity assays was the apparent product inhibition of enzyme activity and the inhibitor may be either peptide and/or glycan components, which require further investigations into the cause of the inhibition