We propose to validate the feasibility of expression in bovine milk of a recombinant protein sweetener, following gene delivery by intramammary infusion of a replication defective viral vector. The sweet protein Brazzein is a natural plant product which has been isolated and characterized and for which the gene construct is available. Brazzein is a protein found in berries of the plant Pentadiplandra brazzeana B. which grows in very focal ecosystems in Central Africa. As a sweetener, Brazzein has many desirable qualities: it is stable at boiling temperature, free of aftertaste, 2000 times as sweet as sucrose, and has no known negative effects on human health. We will insert a simple genetic construct of Brazzein into the mammary cells of several cattle, collect milk from them over an extended period and isolate the protein for evaluation. During Phase II, we will enhance the genetic construct by adding mammary specific control and stabilization sequences to increase the protein yield and will develop pilot plant scale purification methods. Also, in Phase II, we will evaluate the long term production of this protein by cows in their subsequent lactation.Applications:Brazzein is a natural sweetener with a very intense level of sweetness. Its unique properties such as heat stability will enable its use in products and processes which have been incompatible with current sweeteners. When produced in milk it could be used directly, or as a blend, in processed dairy products. This application of recombinant protein technology offers exciting potential for diversification of the dairy industry, as a first of perhaps many options for producing "modified milk" for special contract markets.
