Processing of transgenic corn seed and its effect on the recovery of recombinant β-glucuronidase

• Published in: Biotechnology and bioengineering 1998-10, Vol.60 (1), p.44-52
• Main Authors: Kusnadi, Ann R., Evangelista, Roque L., Hood, Elizabeth E., Howard, John A., Nikolov, Zivko L.
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Animal Feed; Biological and medical sciences; Biotechnology; Catalyst activity; Comminution; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Extraction; Food Supply; Fractionation; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic engineering applications; Genetic technics; Genetics and breeding of economic plants; Glucuronidase - biosynthesis; Glucuronidase - chemistry; Glucuronidase - isolation & purification; Heating; Methods. Procedures. Technologies; Moisture; Paraffins; Plant breeding: fundamental aspects and methodology; Plants, Genetically Modified - enzymology; protein extraction; recombinant enzyme; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; recovery; Seed; Seeds - enzymology; Thermodynamic stability; Transgenic animals and transgenic plants; transgenic corn; Transgenic plants; Zea mays; Zea mays - enzymology; β-glucuronidase
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19981005)60:1<44::AID-BIT5>3.0.CO;2-0

The tools of plant biotechnology that have been developed to improve agronomic traits are now being applied to generate recombinant protein products for the food, feed, and pharmaceutical industry. This study addresses several processing and protein recovery issues that are relevant to utilizing transgenic corn as a protein production system. The gus gene coding for β‐glucuronidase (rGUS) was stably integrated and expressed over four generations. The accumulation level of rGUS reached 0.4% of total extractable protein. Within the kernel, rGUS was preferentially accumulated in the germ even though a constitutive ubiquitin promoter was used to direct gus expression. Fourth‐generation transgenic seed was used to investigate the effect of seed processing on the activity and the recovery of rGUS. Transgenic seed containing rGUS could be stored at an ambient temperature for up to two weeks and for at least three months at 10°C without a significant loss of enzyme activity. rGUS exposed to dry heat was more stable in ground than in whole kernels. The enzyme stability was correlated with the moisture loss of the samples during the heating. Transgenic seed was dry‐milled, fractionated, and hexane extracted to produce full‐fat and defatted germ fractions. The results of the aqueous extraction of rGUS from ground kernels, full‐fat germ, and defatted‐germ samples revealed that approximately 10 times more rGUS per gram of solids could be extracted from the ground full‐fat germ and defatted‐germ than from the kernel samples. The extraction of corn oil from ground germ with hot hexane (60°C) did not affect the extractable rGUS activity. rGUS was purified from ground kernels and full‐fat germ extracts by ion exchange, hydrophobic interaction, and size exclusion chromatography. Similar purity 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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 44–52, 1998.