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 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)
Hong
Y R
YR
Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore 21201-1503, USA.
Mullaney
J M
JM
Black
L W
LW
D017122:Bacteriophage T4 / Q000235:genetics* / Q000254:growth & development; D017123:Bacteriophage T7 / Q000201:enzymology; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D036022:Capsid Proteins; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005822:Genetic Vectors / Q000187:drug effects; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019255:NADPH Oxidases; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014764:Viral Proteins
D036022:Capsid Proteins; D003573:Cytochrome b Group; D015699:HIV Envelope Protein gp120; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C081364:internal protein III, bacteriophage T4; D005914:Globins; C036888:cytochrome b558; D019255:NADPH Oxidases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010450:Endopeptidases