RESUMO
Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.
Assuntos
Proteínas do Leite/genética , Leite Humano/química , Plantas Geneticamente Modificadas/metabolismo , Adulto , Animais , Reatores Biológicos , Caseínas/biossíntese , Bovinos , Humanos , Lactalbumina/biossíntese , Lactoferrina/biossíntese , Proteínas do Leite/biossíntese , Proteínas Recombinantes/biossíntese , Solanum tuberosumRESUMO
A 1177 bp cDNA fragment encoding the human milk protein beta-casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase (mas1',2') promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human beta-casein cDNA. The presence of human beta-casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human beta-casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human beta-casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human beta-casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as beta-casein. The above experiments demonstrate the expression of human milk beta-casein as part of an edible food plant. These findings open the way for reconstitution of human milk in edible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children.
Assuntos
Caseínas/genética , Caseínas/metabolismo , Proteínas Recombinantes/genética , Solanum tuberosum/genética , Caseínas/imunologia , Clonagem Molecular , Vetores Genéticos/genética , Humanos , Plantas Geneticamente Modificadas/genética , Proto-Oncogene Mas , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transformação GenéticaRESUMO
Because previous purification procedures for human kappa-casein may have caused the loss of some carbohydrate, relatively gentle methods were used. The protein was isolated by a four-step procedure which included isoelectric precipitation of whole casein, gel chromatography on Sephadex G-200 in the presence of SDS, removal of the SDS with Extracti-Gel D, and FPLC chromatography on Mono Q with buffers containing 6 M urea. The purified protein was nearly identical in amino acid composition to that found earlier by amino acid analysis and peptide sequencing and a molar extinction coefficient of 11.2 +/- 0.1 was determined on the basis of amino acid analysis with a norleucine internal standard. Hydrolysis, acylation, and methylsilylation of the carbohydrate, followed by gas chromatographic analysis on a fused silica column, yielded approximately 5% fucose, 17% galactose, 18% N-acetylglucosamine, 8% N-acetylgalactosamine and 7% sialic acid, totaling almost 55% by weight. The percentages from two different donors were almost the same. About 1 mole phosphorus per mole of kappa-casein was also detected. Using low-speed sedimentation equilibrium methods, a molecular weight of only 33,400 was obtained for human kappa-casein, suggesting carbohydrate lability. Human beta-casein with four phosphoryls was stabilized against precipitation by 10 mM Ca+2 ions at a level greater than 95% when the molar ratio of kappa/beta exceeded 0.15.