PROCESSING OF TRANSGENIC CORN SEED AND ITS EFFECT ON THE RECOVERY OF RECOMBINANT BETA-GLUCURONIDASE

The tools of plant biotechnology that have been developed to improve a gronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study a ddresses several processing and protein recovery issues that are relev ant to utilizing transgenic corn as a protein production system. The g us gene coding for beta-glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reach ed 0.4% of total extractable protein. Within the kernel, rGUS was pref erentially accumulated in the germ even though a constitutive ubiquiti n promoter was used to direct gus expression. Fourth-generation transg enic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS co uld be stored at an ambient temperature for up to two weeks and for at least three months at 10 degrees C without a significant loss of enzy me activity. rGUS exposed to dry heat was more stable in ground than i n whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry-mille d, fractionated, and hexane extracted to produce full-fat and defatted germ fractions. The results of the aqueous extraction of rGUS from gr ound kernels, full-fat germ, and defatted-germ samples revealed that a pproximately 10 times more rGUS per gram of solids could be extracted from the ground full-fat germ and defatted-germ than from the kernel s amples. The extraction of corn oil from ground germ with hot hexane (6 0 degrees C) did not affect the extractable rGUS activity. rGUS was pu rified from ground kernels and full-fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar p urity and yield of rGUS were obtained from both extracts. Biochemical properties of rGUS purified from transgenic corn seed were similar to those of E. coli GUS. (C) 1998 John Wiley & Sons, Inc.