COMMERCIAL PRODUCTION OF BETA-GLUCURONIDASE (GUS) - A MODEL SYSTEM FOR THE PRODUCTION OF PROTEINS IN PLANTS

We have generated transgenic maize seed containing beta-glucuronidase (GUS) for commercial production. While many other investigators have d emonstrated the expression of GUS as a scoreable marker, this is one o f the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant beta-glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populatio ns of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest lev el of expression. In seeds, the majority of recombinant protein was pr esent in the embryo, and subcellular localization indicated that the p rotein was dispersed throughout the cytoplasm. The purified recombinan t beta-glucuronidase (GUS) was compared to native beta-glucuronidase u sing SDS-PAGE and western blot analysis. The molecular mass of both th e recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predic ted from the cloned Escherichia coil gene except that the initial meth ionine was cleaved from the recombinant GUS. The recombinant and nativ e GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purifi ed proteins were stable for 30 min at 25, 37, and 50 degrees C. Kineti c analysis of the recombinant and native GUS enzymes using 4-methylumb elliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K-m of 0.20 mM and a V-max of 0.29 mM MUG per hour, and the native enz yme had a K-m and V-max of 0.21 mM and 0.22 mM/h respectively. Using D -saccharic acid 1,4-lactone, which is an inhibitor of beta-glucuronida se, the K-i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functio nally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have use d an efficient recovery system where the final protein product, GUS, h as been successfully purified. We describe one of the first model syst ems for the commercial production of a foreign protein which relies on plants as the bioreactor.