Date of Award:
Master of Science (MS)
Animal, Dairy, and Veterinary Sciences
John D. Morrey
There are numerous proteins that have potential uses in commercial and scientific applications that are not utilized to their full potential. this is partly because it is not economically feasible to isolate some of these proteins from their natural sources or to produce them using bacterial fermentation methods. The purpose of this research was to target recombinant protein expression to the mammary glands of genetically engineered or transgenic animals. Foreign protein expression has been achieved in the mammary glands of rabbits, sheep, cows, and swine. By using a strong mammary gland promoter and signal peptide fused to the protein, it was hypothesized at the beginning of the study that the two proteins of this study would be secreted into the milk.
To test this approach for protein production, expression vectors for two different plant proteins were made. The proteins targeted for expression were thaumatin and brazzein, proteins that have sweetener or flavor altering properties. The regulatory portion of the expression vector used exons and introns from the milk β-casein gene. Four and a half kilobases of the 5' region of the bovine β-casein gene was isolated, which contained the promoter sequence and other regulatory sequences for gene expression in mammary tissue. A size of 2.2 kilobases of the 3' region of the β-casein gene contained further regulatory sequences as well as a polyadenylation signal. The gene sequence for the protein was modified by using codons commonly used for casein and was generated using synthetic oligonucleotides. Additionally, the signal peptide from the alpha S-1 casein gene was used to transport the protein into the mammary milk vesicle. The DNA expression vectors were subsequently injected into murine and caprine embryos for the production of transgenic animals. Transgenic mice and a goat were identified that contained the thaumatin transgene. Preliminary analysis of mouse milk by capillary gel electrophloresis indicated the expression of thaumatin protein. This protein expression system is intended to utilize large dairy animals as bioreactors for efficient, non-toxic protein production with a view to being applied to different proteins as the technology advances.
Bates, Katherine M., "Protein Production in the Milk of Genetically Engineered Animals" (1998). All Graduate Theses and Dissertations. 3980.
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