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dc.contributor.authorHong, Yi-Ren*
dc.date.accessioned2012-06-15T20:16:31Z
dc.date.available2012-06-15T20:16:31Z
dc.date.issued1993
dc.identifier.urihttp://hdl.handle.net/10713/1646
dc.descriptionUniversity of Maryland, Baltimore. Ph.D. 1993en_US
dc.description.abstractWe 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.en_US
dc.language.isoen_USen_US
dc.subjectBiology, Molecularen_US
dc.subjectBiology, Geneticsen_US
dc.subjectBiology, Microbiologyen_US
dc.subjectexpression-packaging-processing vectoren_US
dc.subject.meshBacteriophage T4en_US
dc.subject.meshGenetic Vectorsen_US
dc.titleBacteriophage T4 as an expression-packaging-processing vectoren_US
dc.typedissertationen_US
dc.contributor.advisorBlack, Lindsay W.
dc.identifier.ispublishedYes
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