       Document 0492
 DOCN  M9610492
 TI    Protection from proteolysis using a T4::T7-RNAP phage
       expression-packaging-processing system.
 DT    9601
 AU    Hong YR; Mullaney JM; Black LW; Department of Biological Chemistry,
       University of Maryland,; School of Medicine, Baltimore 21201-1503, USA.
 SO    Gene. 1995 Aug 30;162(1):5-11. Unique Identifier : AIDSLINE MED/96009870
 AB    DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted
       into a positive selection-vector form of the T4 genome, placing it under
       the control of bacteriophage T4 ipIII promoters. The recombinant
       T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in
       infected cells. Fusion genes were constructed by insertion into a
       plasmid containing an iPIII (encoding internal protein III) target
       portion and a bacteriophage T7 promoter region. When Escherichia coli
       cells containing the plasmid were infected with the T4::T7-RNAP
       re-phage, the bacteria produced fusion protein at high levels. The newly
       synthesized T4::T7-RNAP re-phage progeny package and process the fusion
       protein into the phage capsid during head morphogenesis. In this paper,
       we demonstrate 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 (human immunodeficiency virus envelope protein gp120 V3
       region) are expressed at high levels by T4::T7-RNAP induction. With
       IPIII::beta Glo, expression-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, thought to be degraded by the lon protease. An
       unstable 20-kDa fragment of the large subunit of human cytochrome b558,
       an integral membrane protein in phagocytes, is subject to proteolytic
       degradation even when produced in the lon-deficient BL21 strain.
       However, upon induction with T4::T7-RNAP re-phage, the 20-kDa protein is
       produced intact.(ABSTRACT TRUNCATED AT 250 WORDS)
 DE    Bacteriophage T4/GROWTH & DEVELOPMENT/*GENETICS  Bacteriophage
       T7/ENZYMOLOGY  Capsid/BIOSYNTHESIS/GENETICS  Comparative Study
       Cytochrome b/BIOSYNTHESIS/GENETICS  *Genetic Vectors/DRUG EFFECTS
       Globin/BIOSYNTHESIS/GENETICS  Human  HIV Envelope Protein
       gp120/BIOSYNTHESIS/GENETICS  Peptide Peptidohydrolases/METABOLISM
       *Protein Processing, Post-Translational  Recombinant Fusion
       Proteins/*BIOSYNTHESIS  RNA Polymerases/GENETICS  Support, U.S. Gov't,
       P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

