NDLI: Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

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Molecular Breeding : Volume 4, Issue 3, June 1998

Molecular Breeding : Volume 4, Issue 2, April 1998

Molecular Breeding : Volume 4, Issue 1, February 1998

Molecular Breeding : Volume 3

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

Content Provider	Springer Nature Link
Author	Witcher, Derrick R. Hood, Elizabeth E. Peterson, Dave Bailey, Michele Bond, Diane Kusnadi, Ann Evangelista, Roque Nikolov, Zivko Wooge, Cynthia Mehigh, Richard Kappel, William Register, James Howard, John A.
Copyright Year	1998
Abstract	We have generated transgenic maize seed containing β-glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of 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 β-glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations 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 level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant β-glucuronidase (GUS) was compared to native β-glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl 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 enzyme 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 β-glucuronidase, the K$_{i}$ of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.
Starting Page	301
Ending Page	312
Page Count	12
File Format	PDF
ISSN	13803743
Journal	Molecular Breeding
Volume Number	4
Issue Number	4
e-ISSN	15729788
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	1998-01-01
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Plant Sciences
Content Type	Text
Resource Type	Article
Subject	Genetics Plant Science Molecular Biology Biotechnology Agronomy and Crop Science

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