Progress 09/01/05 to 08/31/07
Outputs
Accomplishments: Objective 1. Develop maize varieties producing GFP for the optimal expression strategy for recovery. We developed a promoter with transcriptional activity in the endosperm and embryo of maize kernels by combining domains of a promoter with endosperm activity with domains of a promoter with embryo activity and testing the resulting constructs using GFP as a reporter gene. We expect that this promoter will have a high level of tissue specificity. Objective 2 (modified). Biochemical characterization of corn seed-derived rGelatin. 50kD gelatin (rG) was purified from transgenic grain and compared to the same product produced in Pichia. With combinations of mass spectrophotometric identification via tryptic digest, MS-MS characterization of the intact protein, N-terminal sequencing, amino acid analysis, and denaturation temperature we have shown it to be equivalent with some small variation in the degree of proline hydroxylation. Objective 3. Synthesize
genes and transform plants for rG. Two rG gene constructs have been introduced into two different types of maize genotypes, male fertile and male sterile. Molecular analysis (RT-PCR) results indicated that these genes were expressing in maize callus culture. Objective 4. Develop grain fractionation procedures to produce enriched fractions. We used a grain pearler to break corn for degerming using 10-g of corn that enables testing small samples for dry degerming potential. Other devices proved ineffective at this scale. When employing our laboratory 100-g wet milling protocol with rLTB-containing corn, 46-72% of the rLTB protein could be recovered in the fiber fractions when steeping without the normal sulfur dioxide and lactic acid, which will enable easy isolation and little loss in valuable starch and protein fractions. The presence of the acids significantly reduced yields. We identified past approaches to produce fractions from rice, oats, and barley. Objective 5. Couple protein
properties to separation behavior. 3-D (size, isoelectric point, hydrophobicity) characterization of corn extracts and the fractions eluting from ion exchange columns have been obtained. Model building to relate the properties to separation behavior has begun using Principal Component Analysis. Objective 6. Demonstrate applications of the targeting, grain fractionation, and extraction and purification strategies. A process and assay for lipase extraction and purification from transgenic corn endosperm was developed and the results published; likewise results for purification of aprotinin from corn germ were submitted and published. Work is just beginning with the first harvest of transgenic corn expressing GFP in endosperm or germ and a next generation planting to obtain higher expression levels has been made. For development of contained field planting protocols, we followed existing APHIS requirements and added additional measures such as bagging tassels and hand pollination.
Impacts
Our methods are providing for high levels of production of valuable proteins in corn with targeted expression to particular portions of the grain. This targeting is combined with milling procedures we have developed to enrich the protein-containing fractions. Such procedures can be done on-farm to add value and eliminate the need to ship viable genetically modified seed. The fractionation also provides for easier purification by early elimination of the fractions not containing product; those fractions can be used to obtain co-products such as fuel ethanol and biodiesel. Our characterization approach for host proteins will enable design of those purification processes for producing such proteins in alternative hosts. Safe production of the source plant is being enhanced by avoiding accumulation in tissues that will not be harvested and by field procedures that prevent gene migration.
Publications
- Gu, Z. and C. E. Glatz. J. Chromatogr. B, 845: 38-50, 2007. Aqueous two-phase extraction for protein recovery from corn extracts.
- Gu, Z. and C. E. Glatz. Sep. Sci. Technol., 42: 1-19, 2007. Recovery of recombinant dog gastric lipase from corn endosperm extract
- Gu, Z. and C. E. Glatz. Biotechnol. Bioengr., 97, 1159-1169, 2007. A method for three dimensional protein characterization and its application to a complex plant (corn) extract.
- Q. Zhong, L. Xu, C. Zhang, and C. E. Glatz, Appl Microbiol Biotechnol 76:607-613, 2007. Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography.
- Shepherd, C; Scott, PM. 2005. Maize Genetics Conference Abstracts. 47:P80 A Maize Chimeric Promoter Drives High Level GFP Expression in Endosperm and Embryo.
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Progress 09/01/05 to 09/01/06
Outputs
Objective 1. Develop maize varieties producing rGFP using different promoters, targeting sequences and genetic backgrounds for process optimization. rGFP expressing grain has been produced in kg quantities for use in processing studies. rGFP lines were advanced and characterized as to transgene copy number, and level and tissue specificity of expression. The highest levels of GFP were 1% in embryo and 6% in endosperm. A novel promoter was designed to function in both germ and endosperm and used to produce rGFP. Objective 2. Characterization of corn-derived rGelatin. 50kD gelatin was purified from transgenic grain and compared to the same product produced in Pichia. Data from N-terminal sequencing, tryptic peptide sequence analysis, and immuno-recognition established the match of the two products. The measured mass (44088 Da) was within 0.2% of the calculated mass and measurable peptide sequences provide a 64% sequence coverage supporting fidelity of expression. Gel
staining revealed no glycosylation. Objective 3. Synthesize genes, transform, and select plants for rGelatin suitable for capsule manufacturing. The industrial collaborator is to provide the preferred gene. Objective 4. Develop grain fractionation procedures to produce enriched fractions. With a laboratory pearler we achieved degerming giving 77% of the oil in 28% of the mass on a 10-g scale. With this procedure we found that 72% of the rLTB protein could be recovered in the fiber fractions. This was surprising because rLTB was thought to be located within starch granules. We surveyed the literature about methods to use in recovering recombinant proteins from rice, oats, and barley and identified approaches to produce fractions enriched recombinant proteins. We established the feasibility of using tissue-specific rGFP containing grain to monitor fractionation procedures (rather than estimating on the basis of oil content) allowing development of new procedures with improved recovery
of specific tissues for higher yield and purity. Objective 5. Expand the 2-D and 3-D database for protein separations via ion exchange. Based on 2-D profiles collected from native corn extracts, three pH values have been selected to investigate 2-D data for protein separations via ion exchange. Both gradient and step elution have been evaluated, but step elution has been selected to develop the database because it is more suitable for database access. A third (hydrophobic) dimension will enhance the model correlation. Objective 6. Demonstrate applications of the targeting, grain fractionation, and extraction and purification strategies. We carried out hand dissection and mechanical fractionation of grain containing rGFP expressed in germ or endosperm; extraction and purification is planned for the current year. The rGelatin was purified for characterization and that work provides us with data for pursuing a lower-cost purification strategy for production.
Impacts
This work is establishing approaches for the safe and economical production of proteins in corn. On farm aspects are aimed at providing elimination of contamination risk and realization of on-farm value addition. Coupling of proteomics to design of purification processes will benefit a variety of production systems.
Publications
- Vignaux, N., Fox, S.R. and Johnson, L.A. 2006. A 10-g laboratory wet-milling procedure for maize and comparison with larger scale laboratory procedures. J. Cereal Sci. 83(5):482-490.
- Vignaux, N., Octavani, D. and Johnson, L.A. 2004. Efficiencies of different types of dry mills in recovering a fraction rich in recombinant protein expressed in endosperm. Annual Meeting of American Association of Cereal Chemists and the Tortilla Industry Association, San Diego, CA. Sept. 19-22. AACC/TIA Annual Meeting Program Book, Abstract 305, pp 141.
- Vignaux, N. and Johnson, L.A. 2006. Wet milling: An efficient process to recover a recombinant LTB-rich fraction from corn. Annual Meeting of American Association of Cereal Chemists, San Francisco, CA. Sept. 17-20. Program Book, World Grains Summit: Foods and Beverages, Abstract P322, pp 173.
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