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    Bacteriophage T4 as an expression-packaging-processing vector

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    Author
    Hong, Yi-Ren
    Advisor
    Black, Lindsay W.
    Date
    1993
    Type
    dissertation
    
    Metadata
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    Abstract
    We have developed a new expression-packaging-processing vector by using the T4 nonessential capsid scaffold protein IPIII. The ipIII gene was expressed at high level in E. coli from plasmids and was truncated at its C-terminus with a E. coli lacZ' multiple cloning site to permit construction of gene fusions in any reading frame. Infection of the expression plasmid-containing bacteria with bacteriophage mutants deleted for the ipIII gene showed that viable phage encapsidated and proteolytically processed the truncated IPIII proteins. An IPIII-{dollar}\beta{dollar}-galactosidase fusion gene produced fusion proteins of MW 130 and 120 kDa which were packaged within viable phage particles. IPIII-{dollar}\beta{dollar}-globin and IPIII-{dollar}\beta{dollar}-globin-{dollar}\beta{dollar}-galactosidase fusion proteins could also be packaged and processed, demonstrating that a eucaryotic protein can be solubilized as an IPIII fusion. By using polymerase chain reaction (PCR) for site-directed mutagenesis to change {dollar}\sb{lcub}90{rcub}{dollar}glu to {dollar}\sb{lcub}90{rcub}{dollar}lys within the {dollar}\beta{dollar}-globin region, cleavage by T4 protease gp21 was completely abolished, suggesting processing occurs at a gp21 consensus site. Blending and in situ X-gal labelling experiments showed that {dollar}\beta{dollar}-galactosidase was injected into host bacteria along with the DNA, suggesting that the proteins are unfolded when packaged or injected with the highly condensed DNA. E. coli strains carrying mutations in groEL and dnaK (molecular chaperones) were used to test for their effect on {dollar}\beta{dollar}-galactosidase activity following injection, and the E. coli dnaK and groEL functions were found to have a major effect on the kinetics of appearance of {dollar}\beta{dollar}-galactosidase activity. We also have designed a method for inserting foreign DNA into the T4 genome for expression. The T4 e gene coding for lysozyme is required for plaque formation and can be used for positive selection. We demonstrated that recombinant T4 not only contains a ipIII-lacZ fusion gene, but also packages the IPIII-{dollar}\beta{dollar}-galactosidase fusion product inside the T4 head. By using this e gene positive selection method, DNA coding for bacteriophage T7 RNA polymerase (T7 RNAP) was inserted into the T4 genome downstream of the bacteriophage T4 ipIII promoters. The recombinant T4::T7 RNAP phage retained infectivity and expressed T7 RNA polymerase in infected cells. Genes were inserted into a plasmid that contained an IPIII target portion and a bacteriophage T7 promoter region. When E. coli cells were infected with the recombinant T4:: T7 RNAP phage, the bacteria expressed fusion proteins at high level. The newly synthesized T4 packaged and processed the fusion protein into the T4 capsid during head morphogenesis. In the IPIII-{dollar}\beta{dollar}-globin case, the expression-packaging-processing (EPP) occurred following infection with the T4::T7 RNAP phage. In conclusion, the EPP systems may allow stabilization, in vivo processing and easy purification of proteins as well as study of their refolding in bacteria.
    Description
    University of Maryland, Baltimore. Ph.D. 1993
    Keyword
    Biology, Molecular
    Biology, Genetics
    Biology, Microbiology
    expression-packaging-processing vector
    Bacteriophage T4
    Genetic Vectors
    Identifier to cite or link to this item
    http://hdl.handle.net/10713/1646
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    Theses and Dissertations All Schools
    Theses and Dissertations School of Medicine

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