Gene transfer technologies for the production of enzyme and protein reference materials

Abstract

To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes. Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues. By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues. We have used these technologies to produce human γ-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells. Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme. Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts. Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials. We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories.