This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F) is widely used for efficient release of N-glycans form a broad range of protein sources. The enzyme will release high mannose, hybrid and complex N-glycans and chitobiose core ?1,6Fuc substitution has little effect on activity. Although the enzyme is not appreciably active on glycans containing core ?1,3Fuc and some other rare substitutions, most N-glycans studied can be released using this enzyme. It is available form several reputable sources. However, cost, batch to batch purity and variation of specific activity can sometimes be problematic. To gain better control over these factors we have decided to produce the enzyme in-house. The plasmid and enzyme purification scheme were the kind gift of Dr. Patrick Van Roey of the Wadsworth Center, where the enzyme was originally isolated, characterized and crystallized. We have adapted the protocol for lab usage and have purified several batches of enzyme. Enzyme batches have been standardized and tested for residual proteolytic and glycosidase activities. The plasmid bearing the PNGase F insert has been transfected into K-12 Escherichia coli and the strain is maintained in cold storage for use in production of additional enzyme batches.
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