Progress 09/15/03 to 09/30/05
Outputs
Protein recovery and purification are limiting factors making plant-made recombinant proteins (therapeutic proteins and industrial enzymes) successful products. This project focused on developing strategies whereby expression of recombinant proteins in corn can be targeted in seed tissues to simplify processing. For a model recombinant protein system, we developed transgenic plants producing the marker protein Green Fluorescent Protein (GFP) in corn endosperm and embryo. Four constructs were derived from the endosperm specific 22 kDa, 27 kDa, and 19 kDa zein promoters and the embryo specific Glb-1 promoter. Lines producing functional GFP with the expected tissue specificity were identified and advanced in a greenhouse trial. We developed a hybrid promoter combining embryo and endosperm activity. Because it is very active in both major seed tissues, this promoter should enable higher levels of foreign proteins than native promoters. For downscaling dry-milling
fractionation methods, we used a corn containing recombinant dog gastric lipase (useful in treating cystic fibrosis patients) to evaluate a disk attrition mill and simple rollermilling, and compared our results with our previous work using a drum degermer and an impact mill. Using the drum degermer, we were able to recover 72% of the seed oil in 25% of the seed mass. The endosperm mass contained 89% of the recombinant lipase activity in 70% of the seed mass. Wet milling fractionation was evaluated as another procedure to recover a lipase-rich fraction and we discovered that lipase was shear sensitive and inactivated during the grinding steps. We used another model corn containing the heat-labile toxin from E Coli (LTB) in the endosperm with small amount embedded within the starch granules. 90% of the LTB was recovered in 35% of the seed mass, with half of it present in the fine fiber fraction. Selective extraction as the first step in achieving purification after preparation of dry
fractions requires an understanding of the solubility profiles of native corn proteins. We determined the salt and pH dependence of native corn protein solubility for corn and germ fractions. Low contamination with native proteins can be best achieved with the endosperm fraction where aqueous solubility is relatively low. For germ, lower contamination can still be achieved by extracting in acidic conditions. Those principles have been applied to two actual recombinant proteins, aprotinin targeted to germ and lipase targeted to endosperm. An enriched extract was obtained for aprotinin by acidic extraction at low salt concentration. This extract was suitable for purifying by cation exchange. Lipase, in contrast, represents the class of very hydrophobic proteins. Its extraction required surfactants, which enabled increased extraction selectivity by an initial extraction with low surfactant concentration to remove ca. 50% of the native proteins, followed by continued extraction after
surfactant addition to obtain the lipase. Defatting of whole corn meal or isolation of the endosperm fraction enabled reduction of surfactant levels and increased yields. Further size reduction increased extraction rate.
Impacts
The cost of extracting corn to produce therapeutic proteins and industrial enzymes is costly due to low concentration, high water use, and presence of contaminating oil and non-target proteins. The results of this project can be used to reduce the cost of extraction, recovery and purification by targeting expression in easily fractionated grain parts and parts in which purification is eased by reducing contaminants. Some of the fractionation technologies may be suitable to conduct on farm, thereby, reducing the potential for contaminating commodity corn with those containing unapproved events. The technologies developed will immediately be utilized by industrial clients in the proposed Biologic Facility. The transgenic GFP plants will facilitate experiments involving processing and recovery of recombinant proteins from grain by providing a target protein that is detectable with higher sensitivity and accuracy and lower expense and effort than other target proteins.
These lines will also allow us to improve grain-based protein production systems. By selecting for lines with high GFP expression level, breeders will be able to develop new corn lines designed for optimal production of foreign proteins in plants. These lines also allow investigations into the agronomic practices best suited to the production of recombinant proteins in plants. The impact of these plants extends well beyond the scope of this proposal as they enable researchers to perform experiments on seed development and will facilitate discovery of genes involved in seed protein deposition.
Publications
- Vignaux, N., D. Octavani, and L.A. Johnson. 2005. 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.
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Progress 01/01/04 to 12/31/04
Outputs
Protein recovery and purification are limiting factors making plant-made recombinant proteins (therapeutic proteins and industrial enzymes) successful products. This project focuses on developing strategies whereby expression of recombinant proteins in corn can be targeted (i.e., bran, germ, endosperm, within starch granules) to simplify processing. For a model recombinant protein system, we designed genes for producing the marker protein Green Fluorescent Protein (GFP) in corn endosperm and embryo, and used them to develop transgenic plants. Lines producing functional GFP with the expected tissue specificity were identified and advanced. The current generation of seeds contain 88% B73 genome and 12% A188 genome. Greenhouse trials will yield enough grain for small-scale processing and extraction experiments in early 2005, and in the fall 2005 we will have grain for Kg-scale experiments. We developed a hybrid promoter having embryo activity not significantly different
from the strongest embryo specific promoter and has endosperm activity not significantly different from the strongest endosperm specific promoter. Because it is very active in both major seed tissues, this promoter should enable higher levels of foreign proteins than native promoters. For downscaling dry-milling methods to fractionate corn into germ- endosperm- and bran-rich fractions, we evaluated a disk attrition mill and simple rollermilling, and compared our results with our previous work using a drum degermer and an impact mill. The drum degermer produced purer fractions, but was closely followed by the disk attrition mill. Using the drum degermer, we were able to recover 72% of the seed oil in 25% of the seed mass. The endosperm mass contained 89% of the recombinant lipase activity in 70% of the seed mass. We developed a 10-g wet-milling procedure to enable testing different wet fractionation procedures. Selective extraction as the first step in achieving purification after
preparation of dry fractions requires an understanding of the solubility profiles of native corn proteins. We have determined the salt and pH dependence of native corn protein solubility for corn and germ fractions. Low contamination with native proteins can be best achieved with the endosperm fraction where aqueous solubility is relatively low. For germ, lower contamination can still be achieved by extracting in acidic conditions. Those principles have been applied to two actual recombinant proteins: aprotinin targeted to germ and lipase targeted to endosperm. An enriched extract was obtained for aprotinin by acidic extraction at low salt concentration. This extract is suitable for purifying by cation exchange. Lipase, in contrast, represents the class of very hydrophobic proteins. Its extraction requires surfactants, which enabled increased extraction selectivity by an initial extraction with low surfactant concentration to remove ca. 50% of the native proteins, followed by
continued extraction after surfactant addition to obtain the lipase. We will pursue alternative dry fraction mechanical processing to reduce extraction time.
Impacts
The cost of extracting corn to produce therapeutic proteins and industrial enzymes is costly due to low concentration, high water use, and presence of contaminating oil and non-target proteins. This project will reduce the cost of extraction, recovery and purification by targeting expression in easily fractionated grain parts and parts in which purification is eased by reducing contaminants. Some of the fractionation technologies may be suitable to conduct on farm, thereby, reducing the potential for contaminating commodity corn with those containing unapproved events. Information on storage stabilities of enriched fractions is important to know: how quickly the fractions must be processed and how they must be stored to maximize biological activity of the recovered recombinant protein and how additional value can be obtained from processing germ such as into oil for biodiesel. The technologies developed will immediately be utilized by industrial clients in the
proposed Biologic Facility. The 10-g wet-milling procedure will be of use to corn breeders as a selection tool for improved corn hybrids.
Publications
- No publications reported this period
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Progress 09/01/03 to 12/31/03
Outputs
Protein recovery and purification are limiting factors making plant-made recombinant proteins (therapeutic proteins and industrial enzymes) successful commercial products, and processing steps typically represent over one-half of the production cost for recombinant proteins. The purpose of this project is to develop strategies whereby expression of recombinant proteins in corn can be targeted (i.e., bran, germ, endosperm, within starch granules) to simplify downstream processing by making proper matches of the native protein matrix, the extraction method, the separation method, and the characteristics of the recombinant protein. A secondary long-term objective is to use this information to produce technologies and systems for specific proteins of interest to businesses using the proposed Biologics Facility located in the ISU Research Park. The project began September 2003 and we expect a combination of multi-year funding will be required to complete the proposed
long-term objectives. We began to prepare a collection of transgenic maize plants matched for parent properties but with controlled differences in targeting with selected recombinant proteins (green fluorescent protein, GFP) for comparing the relative advantages of each. We cloned five promoters and used them to construct different expression vectors designed to produce GFP in the endosperm, germ or aleurone of the bran. These vectors are currently in the plant transformation pipeline. We began to down-scale dry-milling methods for use on-farm and pilot-plant processing trials have commenced using a disk attrition mill for degerming but samples must be analyzed. Other objectives that have not yet been initiated include: identifying effects of storage conditions of dry-milled fractions on yield and biological activity of target proteins and quality of potential co-products; determining the effects of corn steeping conditions in traditional wet milling and quick germ processing on
biological activity target proteins; and determining extraction kinetics of target proteins.
Impacts
The cost of extracting corn to produce therapeutic proteins and industrial enzymes is costly due to low concentration, high water use, and presence of contaminating oil and non-target proteins. This project will reduce the cost of extraction, recovery and purification by targeting expression in easily fractionated grain parts and parts in which purification is eased by reducing contaminants. Some of the fractionation technologies may be suitable to conduct on farm, thereby, reducing the potential for contaminating commodity corn. The technologies developed will immediately be utilized by industrial clients in the proposed Biologic Facility.
Publications
- No publications reported this period
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