Ar. Kusnadi et al., PROCESSING OF TRANSGENIC CORN SEED AND ITS EFFECT ON THE RECOVERY OF RECOMBINANT BETA-GLUCURONIDASE, Biotechnology and bioengineering, 60(1), 1998, pp. 44-52
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.