• Bacteriophage T4 as an expression-packaging-processing vector

      Hong, Yi-Ren; Black, Lindsay W. (1993)
      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.
    • Co-chaperonin specificity in the folding of the bacteriophage T4 major capsid protein

      Andreadis, Joanne D.; Black, Lindsay W. (1997)
      Bacteriophage T4 is unique among phages in that its growth only requires the Escherichia coli GroEL (cpn60) protein but not the GroES (cpn10) co-chaperonin. Our findings indicate that bacteriophage T4 synthesizes its own co-chaperonin, gp31, which in conjunction with GroEL, is strictly required for the folding of the T4 major capsid protein, gp23. Overexpression work demonstrates that GroEL and gp31 are both necessary and sufficient for the proper folding and oligomerization of the major capsid protein in vivo. Although gp31 and GroES have no significant sequence homology, either on the primary amino acid level or on the predicted secondary structural level, the two proteins appear to be functional homologues. Cryoelectron microscopy of gp31 purified to 90% indicates gp31 forms oligomeric ring structures that are comparable to those formed by GroES. Studies using expression vectors and genetic analysis indicate that although the GroEL-gp31 complex can fold proteins normally folded by GroEL-GroES, the reverse is not true. Thus, even when GroES is overproduced it is unable to mediate chaperonin-assisted folding of the T4 major capsid protein. In addition, co-immunoprecipitation experiments using antisera directed against GroEL and GroES, suggest that GroES protein is functional during T4 infection and able to bind GroEL protein. These studies demonstrate that gp31 is specifically required to fold the T4 major capsid protein because of a difference in co-chaperonin function and not because GroES protein is inactivated or limiting during the course of T4 infection. In addition, co-immunoprecipitation experiments demonstrate that although gp31 is required for the folding of the T4 major capsid protein, gp31 is not required for the binding of the major capsid protein to GroEL. T4bypass31 are mutant phage that can bypass the strict chaperonin requirement for growth. The presence of both the bypass 31-1 and bypass 31-2 mutations in gp23 (BY23) allows the major capsid protein to fold, although less efficiently, in a GroEL-gp31 chaperonin independent mode. The mutations that confer the bypass phenotype have now been sequenced to two specific regions of the gene encoding the major capsid protein which appear to be critical folding sites of the polypeptide as judged by previous genetic analysis. The bypass31-1 mutation is a single missense mutation near the 3 prime end of gene that results in the conversion of Ala-455 to Val-455. The bypass31-2 site, located in the center of gene 23, consists of three independent missense mutations that convert Gly-292 to Ser-292, Val-306 to Ile-306, and Val-307 to Ile-307. Characterization of these mutations by site-directed mutagenesis and genetic studies show that all three mutations at the bypass31-2 site are required for optimal phage growth in the absence of GroEL and gp31, but that Ile-306 and Ile-307 are essential to maintain the bypass phenotype. In addition, all four bypass mutations behave additively at elevated temperatures. Mutational analysis also suggests that the bypass phenotype is inherent to the gp23 polypeptide and is not due to translational pausing. (Abstract shortened by UMI.)
    • The role of portal and terminase proteins in the mechanism of DNA packaging in the dsDNA bacteriophage T4

      Baumann, Richard Gerard; Black, Lindsay W. (2002)
      Several of the double-stranded DNA bacterial viruses have been developed into model systems for the study of basic biological and macromolecular functions. While the DNA packaging processes of bacteriophage have been well studied, the exact mechanism by which the energy derived from nucleotide hydrolysis is converted into a translocation of DNA remains unknown. In phage T4, gp20 serves as the donut-shaped, dodecameric portal protein through which DNA passes into the head, and with the gp16 and gp17 terminase proteins, forms a 'packasome' complex capable of translocating DNA. In this dissertation, two independent experimental approaches are described which investigate the role of the portal protein, gp20 and the terminase protein, gp17 in DNA packaging. To examine the function of the gp20 portal in phage T4, and to test the portal rotation model for DNA packaging, we created and incorporated gp20-fusion proteins into phage. 20-GFP (green fluorescent protein), 20-HOC (T4 highly antigenic outer capsid protein), 20-GFP-HOC, and HOC-20 fusions, when expressed in vivo, complement amber mutant defective infecting virus and produce progeny phage. Normally defective 20am mutant phage (20 am(E481) res12, 20am(N50) res325, and 20 am(B8) res492) make progeny phage in bacteria that express these portal fusions if the fusions are co-assembled with either (1) shortened, co-expressed fusion forms from the expression vector, or (2) shortened, near wild-type sized gp20 forms from infecting 20am(E481) or 20am(B8) phage. Surprisingly, fusions made to either gp20 terminus could function to make progeny phage, despite the varied roles of gp20 during phage maturation. Furthermore, N-terminally fused Hoc-20 protein complemented 20amHocam infecting phage, and analyses demonstrated the Hoc protein was positioned exteriorly and bound to its capsid binding site, essentially tethering gp20 portal in position. The ability of large protein fusions to function in making phage argues against an active, or rotating role for the gp20 portal, and suggests such a mechanism may not operate in the packaging of DNA in T4. To investigate the activities of the large subunit terminase, gp17 (70kDa), the gp17 gene was cloned, and a successful method of protein expression and purification was developed. This new purification is a significant improvement over previous methods, and yields pure, soluble gp17, active in in vitro DNA packaging assays. which show titers of 108 phage per mL of extract. (Abstract shortened by UMI.)