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PubMed · 7557416

Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.

Y R Hong·J M Mullaney·L W Black
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Abstract

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)

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Bacteriophage T4Bacteriophage T7CapsidCapsid ProteinsCytochrome b GroupDNA-Directed RNA PolymerasesEndopeptidasesGenetic VectorsGlobinsHIV Envelope Protein gp120HumansNADPH OxidasesProtein Processing, Post-TranslationalRecombinant Fusion ProteinsViral ProteinsComparative StudyJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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Y R Hong, J M Mullaney, L W Black. 1995-08-30. Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.. https://doi.org/10.1016/0378-1119(95)00327-3

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