The objective of this proposal is to develop an improved baculovirus transfer vector that contains features enabling real time monitoring of protein expression, high level protein production and rapid protein purification from the insect cells. We propose to build this new vector based upon our proprietary pPSC12 transfer vector that already contains the powerful polyhedrin promoter, believed by many to be the most powerful promoter found in nature, and the 61k baculovirus chitinase signal sequence that directs protein through glycosylation and secretion pathways. Four new features can be incorporated into this pPSC12 vector within two months using a straightforward cloning strategy. When completed, the new baculovirus transfer vector will have the following features: 1. The polyhedrin gene promoter; 2. The chitinase signal sequence; 3. A green fluorescent protein (GFP) reporter system for real-time monitoring of protein production; 4. His6 and Strep molecular tags for rapid purification of the expressed protein; 5. Factor Xa cleavage site to facilitate the complete removal of all tags; and 6. A ligation independent cloning system for cloning inserts, independent of their sequence. To demonstrate the power of this new vector, we further propose to express and characterize the hemagglutinin proteins from A/panama/2007/99 (H3N2) influenza virus. The proposed new baculovirus transfer vector will lead to significant improvement of protein production yields and significant reduction of operational cost through eliminating several time-consuming and labor-intensive steps in the manufacture and process development. As one of the primary protein expression systems, the improved insect expression system will be an ideal protein expression system in the war against bioterrorism. The insect cell system offers "speed" and "certainty", that is superior to all other systems since virtually any protein can be expressed in insect cells. Protein Sciences Corporation has demonstrated the power of this system by producing large quantity of cGMP grade recombinant proteins upon short notice in the 1997 Hong Kong "Bird Flu" crisis. Furthermore, the recombinant HA protein that is proposed to be expressed and purified as an example, has the potential to be developed into subunit vaccines and/or diagnostic tools in the event of a terrorist attack involving potentially lethal influenza viruses.
Keywords: BACULOVIRUS, INSECT CELLS, PROTEIN EXPRESSION, HEMAGGLUTININ, INFLUENZA, RECOMBINANT PROTEIN, BIODEFENSE, BIOTERRORISM
