Progress 09/01/06 to 08/31/09
Outputs
OUTPUTS: Alternative promoters and other regulatory sequences, along with RNA interference technology for decreasing production of endogenous seed storage proteins, were evaluated for effectiveness in enhancing expression levels of the transgene. The link between production levels of endogenous and recombinant proteins did not exhibit a predictable relationship for either lines carrying known mutants or random-mated populations. We developed a novel promoter with activity in both the endosperm and germ tissues. By producing proteins in these two tissues, it may be possible to produce higher levels of the desired product. We completed two controlled field releases of LT-B corn using a cytoplasmic male sterile (CMS) maize line and (1) verified the LT-B content in corn kernels and continued the next breeding step to make transgene homozygous and (2) found that ~25% of male sterile corn produced tassels but no viable pollen. Several milling options have been examined for their suitability for a pre-extraction purification step for proteins with different characteristics. The several milling fractions that resulted from each process were characterized as to yield and concentration of the recombinant protein. Much of the r-protein seems to be associated with cell wall fiber regardless of the targeted location. The fine-fiber fraction contained fewer proteins than the endosperm fractions, indicating it would be easier to purify the target protein by enriching the protein by wet milling. Although wet milling was more effective in recovering germ than dry milling, corn steeping conditions must be carefully selected to prevent loss of the native functionality. A modified wet-milling process using a short steeping period with less acid reduced protein destruction. It was possible to fractionate barley, but it was not possible to efficiently fractionate barley into a germ-rich fraction to enable recombinant protein recovery when targeted to the germ. Assessment of the ability of maize to properly produce and assemble a complete triple-helical collagen was completed. Molecular characterization of the collagen determined sequence, required post-translational modifications, and the critical functional property of melting temperature. This work was done after purification of the collagen from early generation plants expressing very low levels. With higher levels, we have achieved lower-cost recovery by precipitation. We initiated work on ultrafiltration as an alternative process with collagen and r-GFP serving as models for high and low molecular weight protein behavior. 3-D characterization of the host proteins from soy, alfalfa and milk proteins was completed to provide comparisons among potential hosts for recombinant protein expression that might provide natural recovery advantages for specific host proteins. Statistical models have been prepared to provide the framework for relating these characteristics to separation behavior and the models have been successfully applied to ion exchange chromatography. Extension of this modeling approach to hydrophobic interaction chromatography was initiated. Results were disseminated in over 20 presentations. PARTICIPANTS: Principal investigators: Charles Glatz (Project Director, Chemical and Biological Engineering) responsible for protein purification; Lawrence Johnson (Director Center for Crops Utilization Research/Food Science and Human Nutrition) responsible for grain fractionation; M. Paul Scott (ARS/Agronomy) responsible for GFP expression design and plant transformation; Kan Wang (Director Plant Transformation Facility/Agronomy) responsible for expression strategies and plant transformation for LTB and collagen. Supervised by Scott were Joan M. Peterson - postdoctoral researcher in Agronomy, Colin T. Shepherd - Ph.D. Student (completed degree) in Agronomy, Megan N. Harvey - M.S. Student (completed degree) in Agronomy. Supervised by Wang were Mr. Xing Xu - graduate student in Plant Transformation Facility; Dr. Lorena Moeller - postdoc research associate, participating in field release and transgenic plant analysis; Ms. Qinglei Gan - research associate in Plant Transformation Facility. Supervised by Johnson were Steve Fox - Technician in Center for Crops Utilization Research; and Ilanko Paraman - postdoc in CCUR pilot plant. Supervised by Glatz were Matt Aspelund and Cheng Zhang (degree completed) - Ph.D. students in Chemical and Biological Engineering; Li Xu (degree completed) and Ying Liu (degree completed) - M.S./M.E. students in CBE; and Jan Seibel - Technician in Chemical and Biological Engineering; Jagan Billikanti (visiting PhD student from University of Canterbury, NZ and O. Alejandro Aguilar (visiting PhD student from Tec de Monterrey) who came to learn methods for characterization of host proteins; five undergraduates doing undergraduate research. Major professors of the two visiting students are Conan Fee (Professor of Chemical Engineering, U. Canterbury, NZ) and Marco Rito-Palomares (Professor of Chemical Engineering and Biotechnology, Tec de Monterrey, MX). Collaborators are Julio Baez and Sheri Ameda of Fibrogen. TARGET AUDIENCES: Companies involved in corn processing will apply the results of this research to glean more value from corn. Thus, processing companies are most direct beneficiaries of this work. It is likely that some portion of the added value will be passed on to corn producers in the form of premium crop prices and consumers in the form of less expensive, safer corn products. Biotechnology/pharmaceutical firms needing higher volume products may consider the use of plant hosts for production of recombinant proteins needed in larger quantities and/or from non-animal sources. Graduate and undergraduate students and postdoctoral associates are given the opportunity to be trained in the areas of crop biotechnology, processing and protein purification. Researchers in the biotechnology area receive new knowledge generated from this project that will enhance the understanding plant biology such as gene expression and regulation and separation of complex extracts. Regulators and legislators can use our research on confined field release of transgenic corn expressing pharmaceuticals and industrial products to establish a strategy that can be used for future large scale production of non-food corn plants in open field. Our data will be communicated to regulators, legislators and other stake holders through this process. PROJECT MODIFICATIONS: For the objective of characterization of host cell proteins of alternative hosts, soy, alfalfa, and milk replaced barley because of the interest of external colleagues in the results with those potential hosts.
Impacts
Our work with promoters and targeting strategies for the r-protein and tools for modifying host cell proteins addresses obtaining high-level expression in the plant with benefits in ease of recovery. While our expression systems are tested with particular product proteins, they are generally applicable. Understanding the relationship between expression of transgenes and endogenous genes will allow breeders to maximize production of transgene products. We find that the best approach is to use recurrent selection for transgene expression. This phase of the work benefited our processing research providing grain containing GFP in either endosperm or embryo. The company Aimsbio was started by one of our graduate students to extend technology developed in this research. We completed the introgression of a vaccine transgene from male-fertile transgenic maize to male-sterile germplasm by conventional breeding allowing us to obtain 100% transgenic seeds from an open-field production using a non-transgenic maize line as the pollinator. 25% of male sterile corn produced tassels but no observable viable pollen, suggesting that the male sterile system can be used for production of r-proteins in corn in open field. The protocols may be applicable to any future large scale r-protein production in corn. The development of grain milling alternatives may enable initial processing steps to occur on-farm, aiding containment of viable seed and farm profitability. Grain fractionation provides 2.5 to 6-fold beneficial enrichment regardless of tissue in which r-protein expression is targeted, improving downstream processing efficiency. Protein-lean co-products could be utilized for fuel ethanol production. Wet milling was more effective (yield and purity considerations) in recovering germ from corn than dry milling, while dry-milling process is cost-effective and suitable for on-farm use. The fractionation facilitates use of the starch and oil fractions in a biorefinery while simplifying purification. Much of this is being done in the context of corn-based collagen because of its GRAS potential and co-product value compatible with corn-based bio-refineries. The corn-based collagen had the desired molecular properties and is cost-effectively recoverable by selective precipitation. We have identified the main remaining host protein contaminant and this protein can be targeted for deletion providing for even easier purification. Ours is the first report of corn-based expression of an assembled helical product with hydroxylation levels providing desired gelation. We are developing processes compatible with two desirable alternatives: (1) integration into corn-based biorefineries, and (2) small-scale, on-farm front-end process steps to add value for farmers and address release concerns. In doing so, we are developing the final protein purification methods that can be applied to separation process development for a variety of ag-based production hosts. Predicting ion exchange separation behavior on the basis of three molecular properties directly from protein mixtures advances the goal of simplifying process development.
Publications
- Zhang, C., S. Fox, L. Johnson, C. E. Glatz, Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen-related proteins. Biotechnol. Prog. In press, 2009. online DOI: 10.1002/btpr.220
- Moeller, L., Taylor-Vokes, R., Fox, S., Gan, Q., Johnson, L., Wang, K. Wet-milling transgenic maize seed for fraction enrichment of recombinant subunit vaccine. Biotechnology Progress (2009), in press
- Paraman, I., Fox, S.R., Glatz, C.E., and Johnson, L.A. 2010. Recovering corn germ enriched in recombinant protein by wet-fractionation. Bioresource Technology 101(1): 239-244. doi:10.1016/j.biortech.2009.08.023
- Moeller, L., Gan, Qinglei, Wang, K. A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway. Journal of Experimental Botany 60: 3337-3352(2009)
- Xu, L,. and C. E. Glatz. 2009. Predicting protein retention time in ion-exchange chromatography based on three-dimensional protein characterizations. J. Chromatogr. A,1216: 274-280.
- Zhang, C., J. Baez, C. E. Glatz. Purification and characterization of a 44 kDa recombinant collagen I alpha 1 fragment from corn grain. J. Agric. Food Chem. 2009, 57 880-887.
- Zhang, C., K. K. W. Pappu, J. Baez, C. E. Glatz. Purification and Characterization of a Transgenic Corn Grain-Derived Recombinant Collagen Type I alpha 1. Biotechnol. Prog. In press, 2009. on-line DOI: 10.1002/btpr.257
- Shepherd, C.T., and M.P. Scott. 2009. Construction and evaluation of a maize chimeric promoter with activity in kernel endosperm and embryo. Biotechnology and Applied Biochemistry 52:233-243. (This paper was highlighted the issue of Biotechnology and Applied Biochemistry in which it appeared)
- Wang, K., Frame, B., Xu, X., Moeller, L., Lamkey, K., Wise, R. 2009. Strategies for the production of maize-derived pharmaceuticals using cytoplasm male sterile lines: in vitro tissue culture/transformation and field breeding approaches. Maydica (2009), in press.
- Paraman, I., Vignaux, N., Moeller, L., Fox, S.R., Wang, K., Glatz, C., and Johnson, L. 2009. Recovering fractions of corn enriched in recombinant proteins. AACC International Annual Meeting, Baltimore, MD. Cereal Foods World 54(4):A59.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: We have produced transgenic corn lines carrying two recombinant gelatin gene constructs (100 kD and C1a1). Promoters and other regulatory sequences are being developed to enhance the expression level. We also are attempting to increase levels by decreasing production of endogenous seed storage proteins via RNA interference technology. We found that the link between production levels of endogenous and recombinant proteins (maize lines producing GFP) did not exhibit a predictable relationship either for lines carrying known mutants or for random-mated populations. We have completed another controlled field release of LT-B corn using a cytoplasmic male sterile (CMS) maize line. ~25% of male sterile corn produced tassels but no viable pollen, suggesting that the male sterile system can be used for open field protein production. Several milling options have been examined for their suitability for a pre-extraction purification step for proteins with different characteristics. Corn wet fractionation processes, including quick germ and traditional wet milling, were evaluated as means of recovering fractions enriched in recombinant human collagen that accumulated in corn germ. The several milling that resulted from each process were characterized as to yield and concentration of the recombinant protein. Influence of transgenic kernels physical properties on the recovery of the protein-rich targets were also determined. Lab procedures to wet mill rice were developed to fractionate rice grains, but no procedure for producing suitable fractions of germ to recover transgenic proteins could be identified. Extreme alkali was required to obtain adequate separation and the alkali destroyed biological activities of most target proteins. It was possible to fractionate barley, but it was not possible to efficiently fractionate barley into a germ-rich fraction to enable recombinant protein recovery when targeted to the germ. The small size of germ also makes barley an inefficient crop in which to produce recombinant proteins with targeted expression to germ. Assessment of the ability of maize to properly produce and assemble a complete triple-helical collagen was completed. Molecular characterization of the collagen determined sequence, required post-translational modifications, and the critical functional property of melting temperature. This work was done after purification of the collagen from early generation plants expressing very low levels. By spiking higher levels of collagen into corn extracts we have done an initial assessment of lower-cost recovery by precipitation. And we have initiated work on ultrafiltration as an alternative lower-cost process using spiked collagen and recombinant GFP to serve as models for high and low molecular weight protein behavior in this size-based separation. The feasibility of our 3-D protein characterization was demonstrated for purification by ion exchange chromatography using a set of model proteins. Results of these activities have been disseminated in presentations at a variety of national technical meetings and regular conference calls with our collaborator at FibroGen. PARTICIPANTS: Principal investigators: Charles Glatz (Project Director, Chemical and Biological Engineering) responsible for protein purification; Lawrence Johnson (Director Center for Crops Utilization Research/Food Science and Human Nutrition) responsible for grain fractionation; M. Paul Scott (ARS/Agronomy) responsible for GFP expression design and plant transformation; Kan Wang (Director Plant Transformation Facility/Agronomy) responsible for expression strategies and plant transformation for LTB and collagen. Supervised by Scott is Anastasia Bodnar - graduate student in Agronomy Supervised by Wang are Mr. Xing Xu - graduate student in Plant Transformation Facility and Ms. Qinglei Gan - research associate in Plant Transformation Facility. Supervised by Johnson is Steve Fox - Technician in Center for Crops Utilization Research. Supervised by Glatz are Matt Aspelund, Li Xu, and Cheng Zhang - Ph.D. students in Chemical and Biological Engineering. Collaborators are Julio Baez and Sheri Ameda of Fibrogen. TARGET AUDIENCES: Companies involved in corn processing will apply the results of this research to glean more value from corn. Thus, processing companies are most direct beneficiaries of this work. It is likely that some portion of the added value will be passed on to corn producers in the form of premium crop prices and consumers in the form of less expensive, safer corn products. Biotechnology/pharmaceutical considering the use of plant hosts for production of recombinant proteins needed in larger quantities and/or from non-animal sources. Graduate students are given the opportunity to be trained in the areas of crop biotechnology, processing and protein purification. Researchers in the biotechnology area receive new knowledge generated from this project that will enhance the understanding plant biology such as gene expression and regulation and separation of complex extracts. Regulators and legislators can use our research on confined field release of transgenic corn expressing pharmaceuticals and industrial products to establish a strategy that can be used for future large scale production of non-food corn plants in open field. Our data will be communicated to regulators, legislators and other stake holders through this process. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work at the molecular level with promoters and targeting strategies for the recombinant protein and tools for modifying host cell proteins addresses obtaining high-level expression in the plant with benefits in ease of recovery. While our expression systems are tested with particular product proteins, they will have applicability to a wide range of such protein products. Understanding the relationship between expression of transgenes and endogenous genes will allow breeders to maximize production of transgene products in transgenic plants. Our results suggest that the best approach to designing transgenic plants with high levels of transgene expression is to use recurrent selection for transgene expression rather than to rely on mutants or to develop lines for transgene expression prior to transformation. An immediate benefit is to our processing research as our improvement transgenic maize lines containing GFP in endosperm or embryo has provided sufficient grain for use in the processing studies for the coming year. The company Aimsbio was started by one of our graduate students to apply technology developed in this research to develop novel strategies for producing foreign proteins in plants. The agronomic strategy of developing male-sterile varieties is to minimize the transgenic pollen dispersion in open fields to provide a widely acceptable means of introducing transgenic crops for protein production. The development of grain milling alternatives may enable initial processing steps to occur on-farm, aiding containment of viable seed and farm profitability. The fractionation facilitates use of the starch and oil fractions in a biorefinery while simplifying purification. Much of this is being done in the context of corn-based collagen because of its GRAS potential, potential co-product value compatible with corn-based bio-refineries, and representation of a class of protein-based biomaterials with potentially high market volume. The corn-based collagen had the desired molecular properties and looks to be cost-effectively recoverable by selective precipitation. Outcomes of our milling tests confirmed that the quick germ method recovered protein-enriched germ with much less denaturation and adequate germ yields. The corn grain fractionation procedure enriched the recombinant protein levels to 5- to 6-fold higher than that of unfractionated kernels. The physicochemical characteristics of the transgenic kernels differed from standard kernels, this didn't affect germ recovery. Employing ultrasonics enhanced rice wet milling starch and protein recoveries, but satisfactory germ recovery was not possible. Nor was it possible to recover a separate germ-rich fraction from barley.
Publications
- Shepherd, C.T., N. Vignaux, J.M. Peterson, L.A. Johnson, and M.P. Scott. 2008. Green Florescent Protein as a Tissue Marker in Transgenic Maize Seed. Cereal Chem. 85(2):188-195.
- Shepherd, C.T., N. Vignaux, J.M. Peterson, M.P. Scott, and L. Johnson. 2008. Dry-milling and Fractionation of Transgenic Maize Seed Tissues with Green Florescent Protein as a Tissue Marker. Cereal Chem. 85(2):196-201. (Featured in Cereal Chemistry Research Update #11 and Agricultural Research Service News and Events, September 25, 2008 )
- Bicar, E., H., W. Woodman-Clikeman, V. Sangtong, J. Peterson, M., S.S. Yang, M. Lee, and M.P. Scott. 2008. Transgenic maize endosperm containing a milk protein has improved amino acid balance. Transgenic Research 17:59-71.
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: We have successfully completed another controlled field release of LT-B corn using a cytoplasmic male sterile (CMS) maize line. We are verifyING the LT-B content in corn kernels and continuing the next breeding step to make transgene homozygous. Two gene constructs for collagen were introduced into two different types of maize genotypes, male fertile and male sterile. Preliminary molecular analysis (RT-PCR) of expression in maize callus culture was carried out. Currently, we are in a process of promoter cloning and characterization of different promoters and other regulatory sequences to enhance the protein production. Three different transgenic grains, expressing LTB, GFP and collagen/gelatin were milled. Using the 10-g degerming protocol developed in previous years, we recovered 65% of the oil (representing germ where rGelatin, C1A1, resides) in 25% of the mass; this fraction also contained 28% of the protein in the grain. We have also used the procedure to dry mill corn
expressing rGFP in germ, corn expressing rGFP in endosperm and the inbred parents B73. The fractions are being analyzed for rGFP to determine mass balances and to develop easy measures of dry-milling efficiency by merely measuring fluorescence to determine amounts of non-target tissues contaminating the separated fractions. We confirmed in a second set of wet-milling trials that we recover most of the rLTB protein in fiber fractions from rLTB-containing corn when employing our laboratory 100-g wet milling protocol despite prior evidence indicated rLTB was located within starch granules. Comparisons of wet and dry milling fractionation to provide traditional and recombinant protein products have also been carried out for corn expressing secreted gelatin and intracellularly retained collagen in the germ fraction. We wet milled in the presence and absence of the normal sulfur dioxide and lactic acid at both 37 and 50 C. LTB-containing corn has been dry milled and the fractions are being
analyzed for LTB contents. We will use these fractions for our proteomics study. Two versions of recombinant gelatin/collagen have been purified from early generation/low expression level corn grain by a procedure we developed so that they can be characterized for veracity of expression and, in the case of collagen, proper triple helix formation. Characterization of the gelatin is complete and the expected composition verified by several methods; collagen structure and composition is partly completed. 3-D characterization of the host proteins from soy and milk proteins has been initiated by training visiting students working with those hosts to provide comparisons among potential agricultural hosts for recombinant protein expression that might provide natural recovery advantages for specific host proteins. Statistical models have been prepared to provide the framework for relating these characteristics to separation behavior and the models have been applied to preliminary data on ion
exchange chromatography. Results of these activities have been disseminated in presentations at technical meetings and regular conference calls with our collaborator at FibroGen.
PARTICIPANTS: Principal investigators: Charles Glatz (Project Director, Chemical and Biological Engineering) responsible for protein purification; Lawrence Johnson (Director Center for Crops Utilization Research/Food Science and Human Nutrition) responsible for grain fractionation; M. Paul Scott (ARS/Agronomy) responsible for GFP expression design and plant transformation; Kan Wang (Director Plant Transformation Facility/Agronomy) responsible for expression strategies and plant transformation for LTB and collagen. Supervised by Scott are Joan M. Peterson - postdoctoral researcher in Agronomy, Colin T. Shepherd - Ph.D. Student (completed degree) in Agronomy, Megan N. Harvey - M.S. Student (completed degree) in Agronomy. Supervised by Wang are Mr. Xing Xu - graduate student in Plant Transformation Facility and Ms. Qinglei Gan - research associate in Plant Transformation Facility. Supervised by Johnson is Steve Fox - Technician in Center for Crops Utilization Research. Supervised by
Glatz are Matt Aspelund, Li Xu, Ying Liu, and Cheng Zhang - Ph.D. students in Chemical and Biological Engineering; Jan Seibel - Technician in Chemical and Biological Engineering; Jagan Billikanti (visiting PhD student from University of Canterbury and O. Alejandro Aguilar (visiting PhD student from Tec de Monterrey) who came to learn methods for characterization of host proteins. Major professors of the two visiting students are Conan Fee (Professor of Chemical Engineering, U. Canterbury, NZ) and Marco Rito-Palomares (Professor of Chemical Engineering and Biotechnology, Tec de Monterrey, MX). Dr. Julio Baez is our collaborator at FibroGen, Inc., S. San Francisco, CA.
TARGET AUDIENCES: Companies involved in corn processing will apply the results of this research to glean more value from corn. Thus, processing companies are most direct beneficiaries of this work. It is likely that some portion of the added value will be passed on to corn producers in the form of premium crop prices and consumers in the form of less expensive, safer corn products. Biotechnology/pharmaceutical considering the use of plant hosts for production of recombinant proteins needed in larger quantities and/or from non-animal sources. Graduate students are given the opportunity to be trained in the areas of crop biotechnology, processing and protein purification. Researchers in the biotechnology area receive new knowledge generated from this project that will enhance the understanding plant biology such as gene expression and regulation and separation of complex extracts. Regulators and legislators can use our research on confined field release of transgenic corn expressing
pharmaceuticals and industrial products to establish a strategy that can be used for future large scale production of non-food corn plants in open field. Our data will be communicated to regulators, legislators and other stake holders through this process.
Impacts
We improved transgenic maize lines containing GFP in endosperm or embryo and produced quantitites of each of these providing sufficient grain for use in the processing studies for the coming year. The company Aimsbio was started by one of our graduate students to apply technology developed in this research to develop novel strategies for producing foreign proteins in plants. Field release of transgenic corn expressing pharmaceutical gene introduced to a male sterile corn variety is a strategy is to minimize the transgenic pollen disperse in open field. The final product of this breeding process will be male sterile pharmaceutical corn that can be grown in the open field and produce 100% seeds expressing pharmaceutical gene. The protocol and Standard Operation Procedure (SOP) established for this project may be applicable to any future large scale non-food production using corn as factory. It is important that we establish trust with state regulators, legislators and
other stake holders through this process with our sound scientific approach and transparent communication. Two gene constructs for collagen, designated as 100 kD and C1a1, have been introduced into two different types of maize genotypes, male fertile and male sterile. Preliminary molecular analysis (RT-PCR) results indicated that these genes were expressing in maize callus culture. The purpose of the corn-based collagen project is to generate transgenic corn plants expressing high level of collagen in seed as a higher value co-product compatible with corn-based refineries. Thus processing of transgenic grain for co-product recovery is aimed at developing processes that yield a high-valued protein product in addition to the traditional oil and starch products. Outcomes at the early milling steps (using several transgenic grains) have confirmed in wet-milling trials that we recover most of the rLTB protein in fiber fractions from rLTB-containing corn when employing our laboratory 100-g
wet milling protocol despite prior evidence indicated rLTB was located within starch granules. Steeping of rCollagen grain in the presence of sulfur dioxide and lactic acid at 37 C yielded less starch and more fiber at 37 C than at 50 C. The germ recovered when steeping at 50 C in the presence of sulfur dioxide and lactic acid yielded a germ fraction with 15-fold higher levels of collagen than in the whole corn whereas steeping at 37 C produced only 7-fold higher concentrations. The collagen remained with the germ fraction for both wet and dry milling while the gelatin was only retained in that fraction for dry milling. We are developing process compatible with two desirable alternatives: (1) integration into corn-based biorefineries, and (2) small-scale, on-farm front-end process steps to add value for farmers and address release concerns. In doing so, we are developing the final protein purification methods that can be applied to separation process development for a variety of
ag-based production hosts with collaborators at two different international universities applying our methods to hosts being studied in their laboratories.
Publications
- Scott, M.P., J.M. Peterson, D.L. Moran, V. Sangtong, and L. Smith. 2007. A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability. Transgenic Research 16:629-643.
- Bicar, E., H. , W. Woodman-Clikeman, V. Sangtong, J. Peterson, M. , S.S. Yang, M. Lee, and M.P. Scott. 2007. Transgenic maize endosperm containing a milk protein has improved amino acid balance. Transgenic Research 11:11-20.
- Jia, H., D. Nettleton, J.M. Peterson, G. Vazquez-Carrillo, J.-L. Jannink, and M.P. Scott. 2007. Comparison of Transcript Profiles in Wild-Type and o2 Maize Endosperm in Different Genetic Backgrounds. Crop Science 47:S-45-59.
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