PROTECTION FROM PROTEOLYSIS USING A T4 /T7-RNAP PHAGE EXPRESSION-PACKAGING-PROCESSING SYSTEM/

DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it und er the control of bacteriophage T4 ipIII promoters. The recombinant T4 ::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in in fected cells. Fusion genes were constructed by insertion into a plasmi d containing an iPIII (encoding internal protein III) target portion a nd a bacteriophage T7 promoter region. When Escherichia coli cells con taining the plasmid were infected with the T4::T7-RNAP re-phage, the b acteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein in to the phage capsid during head morphogenesis. In this paper, we demon strate that truncated T4 internal protein IPIII, human IPIII::beta Glo (beta-globin) fusion protein, E. coli IPIII::beta Glo::beta Gal (beta -galactosidase) triple-fusion protein and IPIII::V3 fusion protein (hu man immunodeficiency virus envelope protein gp120 V3 region) are expre ssed at high levels by T4::T7-RNAP induction. With IPIII::beta Glo, ex pression-packaging-processing (EPP) occurs simultaneously with T4::T7- RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of beta Gal, tho ught to be degraded by the lon protease. An unstable 20-kDa fragment o f the large subunit of human cytochrome b(558), an integral membrane p rotein in phagocytes, is subject to proteolytic degradation even when produced in the lon-deficient BL21 strain. However, upon induction wit h T4::T7-RNAP re-phage, the 20-kDa protein is produced intact. Thus, T 4::T7-RNAP re-phage appear to provide a simple, rapid and universal me ans of producing proteins in high yield, packaging and processing IPII I fusion proteins into easily manipulated phage capsids, and protectin g proteins from proteolysis.