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L W Black

Publications and source records attributed to L W Black.

At least 19 recordsLinked to original sources
PubMed2000

Green fluorescent protein as a probe of rotational mobility within bacteriophage T4.

Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment. Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant phage T4[CTS/IPIII/GFP], T4[CTS/IPIII(T)GFP], and T4[CTS/GFP] packaged GFP fusion proteins and processed them at cleavage sites designated /. Steady-state and time-resolved fluorescence measurements suggest that packaged GFP is concentrated to a high density, that fusion protein IPIII(T)GFP occurs in a tightly clustered arrangement, and that the internal milieu of the phage head reduces rotational mobility of GFP. Phage, but not proheads, packaged with fusion protein IPIII(T)GFP gave an unexpectedly lower anisotropy than phage and proheads packaged with GFP, which suggests IPIII(T)GFP is bound to DNA in a manner that causes close associations between GFP molecules resulting in homotransfer between fluorophores within packaged phage. Targeting of reporter proteins into active virions is a promising approach for determining the structure of the condensed DNA, and properties of encapsidated viral enzymes.

Bacteriophage T4↗
PubMed1999

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda.

Amino Acid Sequence↗
PubMed1998

Substrate mutations that bypass a specific Cpn10 chaperonin requirement for protein folding.

The bacteriophage T4 GroES homologue, gp31, in conjunction with the Escherichia coli chaperonin GroEL, is both necessary and sufficient to fold the T4 major capsid protein, gp23, to a state competent for capsid assembly as shown by in vivo expression studies. GroES is unable to function in this role as a productive co-chaperonin. The sequencing and characterization of mutations within gp23 that confer GroEL and gp31 chaperonin-independent folding of the mutant protein suggest that the chaperonin requirements are due to specific sequence determinants or structures in critical regions of gp23 that behave in an additive fashion to confer a chaperonin bypass phenotype. Conservative amino acid substitutions in these critical regions enable gp23 to fold in a GroEL-gp31 chaperonin-independent mode, albeit less efficiently than wild type, both in vivo and in vitro. Although the presence of functional GroEL-gp31 enhances folding of the mutated gp23 in vivo, GroEL-GroES has no such effect. Site-directed mutagenesis experiments suggest that a translational pausing mechanism is not responsible for the bypass mutant phenotype. Polyhead reassembly experiments are also consistent with direct, post-translational effects of the bypass mutations on polypeptide folding. Given our finding that gp31 is not required for the binding of the major capsid protein to GroEL and that active GroES is incapable of folding the gp23 polypeptide chain to native conformation, our results suggest co-chaperonin specificity in the folding of certain substrates.

Amino Acid Substitution↗
PubMed1998

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range </=10 to 200 molecules of SN packaged per head, and independent of proteolytic cleavage of SN from the IPIII portion of the fusion, favoring a discontinuous packaged DNA structure. Rods of B-form DNA could be envisioned as protected from digestion, whereas bent or kinked DNA would be more susceptible to the diffusible SN. Such discontinuous packaged DNA structures are favored for phage T4 by a number of lines of evidence.

Bacteriophage T4↗
PubMed1998

Phage T4 SOC and HOC display of biologically active, full-length proteins on the viral capsid.

The T4 phage capsid accessory protein genes soc and hoc have recently been developed for display of peptides and protein domains at high copy number (Ren et al., 1996. Protein Science 5, 1833-1843; Ren et al., 1997. Gene 195, 303-311). That biologically active and full-length foreign proteins can be displayed by fusion to SOC and HOC on the T4 capsid is demonstrated in this report. A 271-residue heavy and light chain fused IgG anti-EWL (egg white lysozyme) antibody was displayed in active form attached to the COOH-terminus of the SOC capsid protein, as demonstrated by lysozyme-agarose affinity chromatography (>100-fold increase in specific titer). HOC with NH2-terminal fused HIV-I CD4 receptor of 183 amino acids can be detected on the T4 outer capsid surface with human CD4 domain 1 and 2 monoclonal antibodies. The number of molecules of each protein (10-40) bound per phage and their activity suggest that proteins can fold to native conformation and be displayed by HOC and SOC to allow binding and protein-protein interactions on the capsid.

Bacteriophage T4↗
PubMed1998

DNA requirements in vivo for phage T4 packaging.

Phage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 terminase gene sequences were introduced into prophage lambda imm434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles. Multiple copy prophage lambda imm434 genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total. Deletion or base change of the 24-bp gene 16 3' region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac sequence. Moreover, a specific gene 16-containing pac fragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion. We conclude that both the copy number of homologous sequences and the DNA pac sequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4.

Bacteriophage T4↗
PubMed1998

GFP:HIV-1 protease production and packaging with a T4 phage expression-packaging processing system.

A bacteriophage T4-derived protein expression, packaging and processing system was used to create recombinant phage that encode, produce and package a protein composed of human HIV-1 protease fused to green fluorescent protein (GFP). The fusion protein is targeted within the phage capsid by an N-terminal capsid targeting sequence (CTS), which is cleaved through proteolysis by the viral scaffold protease P21. The fusion protein is designated CTS [symbol see text] GFP:PR. The [symbol see text] symbol indicates the linkage peptide sequence leu(ile)-N-glu that is cleaved by the T4 head morphogenetic proteinase gp21 during head maturation. The fusion protein is fluorescent and has protease activity as detected by the appearance of the expected substrate cleavage product on a Western blot. CTS [symbol see text] GFP:PR packaging occurs at about 200 molecules per phage particle. The CTS [symbol see text] GFP:PR fusion protein, when protected within the phage capsid, has been maintained stably for over 16 months at 4 degrees C. Production and storage of fusion protein within the phage circumvents problems of toxicity and solubility encountered with E. coli expression systems. Because recombinant phage inhibit host proteolytic enzymes, foreign proteins are stabilized. This phage system packages and processes the fusion protein by means of the CTS. Proteins can be purified from the phage to give high yields of soluble, proteolytically processed protein. The T4 phage packaging system provides a novel means of identification, purification and long-term storage of toxic proteins whose folding and DNA-directed activities can be studied readily in vivo.

Bacteriophage T4↗
PubMed1997

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.

Capsid↗
PubMed1997

Purification and characterization of the small subunit of phage T4 terminase, gp16, required for DNA packaging.

Phage T4 terminase is an enzyme that binds to the portal protein of proheads and cuts and packages concatemeric DNA. The T4 terminase is composed of two subunits, gene products (gp) 16 and 17. The role of the small subunit, gp16, in T4 DNA packaging is not well characterized. We developed a new purification procedure to obtain large quantities of purified gp16 from an overexpression vector. The pure protein is found in two molecular weight forms, due to specific C-terminal truncation, displays in vitro packaging activity, and binds but does not hydrolyze ATP. gp16 forms specific oligomers, rings, and side-by-side double rings, as judged by native polyacrylamide gel electrophoresis and scanning transmission electron microscopy measurements. The single ring contains about eight monomers, and the rings have a diameter of about 8 nm with a central hole of about 2 nm. A DNA-binding helix-turn-helix motif close to the N terminus of gp16 is predicted. The oligomers do not bind to DNA, but following denaturation and renaturation in the presence of DNA, binding can be demonstrated by gel shift and filter binding assays. gp16 binds to double-stranded DNA but not single-stranded DNA, and appears to bind preferentially to a gene 16-containing DNA sequence.

Adenosine Triphosphatases↗
PubMed1996

Capsid targeting sequence targets foreign proteins into bacteriophage T4 and permits proteolytic processing.

A membrane-independent morphogenetic viral signal peptide is identified within bacteriophage T4 internal protein III (IPIII). Utilizing a phagederived expression-packaging-processing system, which packages foreign proteins fused with IPIII into the phage capsid, a synthetic cleavage site introduced at the C terminus of IPIII, is demonstrated to be functional and permits processing of fusion proteins. IPIII, which possesses a native P21 cleavage site at its N terminus, is altered to possess a second P21 cleavage site at its C terminus where cleavage occurs by means of the scaffold proteinase P21 within the capsid. The altered IPIII was inserted into an expression vector to permit the creation of fusion proteins with staphylococcal nuclease, EcoRI endonuclease, beta-globin, and luciferase. Western immunoblot analysis of packaged T4eG326 indicates that the IPIII:fusion-proteins are packaged into phage and proteolytically processed, thus the synthetic P21 cleavage site positioned at the C terminus of IPIII is demonstrated to be functional, and 20 to 200 protein molecules are packaged per capsid. Truncation experiments identified the minimal portion of IPIII required to achieve targeting into the phage capsid as a ten amino acid residue from the N terminus, which includes the N-terminal methionine residue and the proteinase P21 cleavage site, designated the CTS (capsid targeting sequence). The addition of the CTS to a fragment of luciferase permits the protein to be packaged and processed, which demonstrates that the CTS is by itself sufficient to target foreign protein to the capsid. The imputed dual function of the CTS is supported by site-directed PCR mutagenesis, which reveals two functionally separate domains of the CTS for targeting and processing. The CTS appears to function in a core-related targeting mechanism that directs a polymorphic set of proteins into the T-even capsid or scaffold. Although structure formation is often assumed to involve extended protein interfaces, the analysis shows that a limited but specific sequence, the CTS, drives the interaction required to achieve targeting.

Amino Acid Sequence↗
PubMed1996

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.

Animals↗
PubMed1995

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)

Bacteriophage T4↗
PubMed1995

Bacteriophage T4 gene 17 amplification mutants: evidence for initiation by the T4 terminase subunit gp16.

Bacteriophage T4 genes 16 and 19 containing the 24 bp homology regions that recombine to form Hp17 mutants were cloned into plasmids. When the two homology sequences were cloned either together into one or separately into two compatible plasmids, a polymerase chain reaction assay showed that recombination occurred in vivo. The recombinant sequence was identical with that found in T4 phage Hp17 mutants, and was produced in recombination-deficient Escherichia coli. Mutational analysis revealed a requirement for functional gene 16 but not gene 17 to recombine the sequences. Moreover, gp16, the terminase small subunit, was required, since an amber gene 16 produced the recombinant sequence only when suppressed. Mutations in the gene 16 recombination sequence (3GA and 15TG) that eliminated Hp17 formation in T4 phage increased the synthesis of the large terminase subunit, gp17 in T4 infections, suggesting gp16 interaction with this site. gp16 binding to gene 16 and gene 19 pac-like sites may synapse the homologous sequences to lead to Hp17 mutant formation, and this suggests a synapsis mechanism for control of T4 DNA maturation and concatemer processing in packaging.

Bacteriophage T4↗
PubMed1995

Mutational analysis of the sequence-specific recombination box for amplification of gene 17 of bacteriophage T4.

Bacteriophage T4 gene 17 amplification mutants Hp17 that carry two to six tandem repeats of the genes 17-18 region were isolated by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli. These mutants arise from an initial sequence-specific recombination between two GCTCA sequences in a 24 bp imperfect homology box in genes 16 and 19. The initial recombination occurred in the wild-type phage T4 population, as shown by polymerase chain reaction, at a frequency of about 10(-6), which is consistent with the frequency of mutant isolation. T4 phage with mutations of the 3rd, 6th, 9th, 12th, or 15th positions in the 24 bp box of gene 16 either failed to produce gene amplification mutant Hp17 or produced gene amplification mutants from an initial recombination at other regions. Among the mutants that failed to produce gene amplification mutants, the initial recombination generally occurred at lower frequencies at either the GCTCA sequence or other sequences. Since the gene amplification mutations are eliminated or shifted to different sequences by base changes that increase as well as decrease homology, the predominant recombination event between the gene 16 and 19 recombination boxes appears to be sequence-dependent rather than homology-dependent.

Amino Acid Sequence↗
PubMed1995

DNA packaging and cutting by phage terminases: control in phage T4 by a synaptic mechanism.

Phage DNA packaging occurs by DNA translocation into a prohead. Terminases are enzymes which initiate DNA packaging by cutting the DNA concatemer, and they are closely fitted structurally to the portal vertex of the prohead to form a 'packasome'. Analysis among a number of phages supports an active role of the terminases in coupling ATP hydrolysis to DNA translocation through the portal. In phage T4 the small terminase subunit promotes a sequence-specific terminase gene amplification within the chromosome. This link between recombination and packaging suggests a DNA synapsis mechanism by the terminase to control packaging initiation, formally homologous to eukaryotic chromosome segregation.

Bacteriophage T4↗
PubMed1993

An expression-packaging-processing vector which selects and maintains 7-kb DNA inserts in the blue T4 phage genome.

We have developed an efficient positive-selection vector to insert foreign DNA segments fused to the T4 ipIII gene (encoding internal protein IPIII) into the bacteriophage T4 genome. By using partial deletions of the T4 e gene, which encodes phage lysozyme, lysozyme activity required for plaque formation is used to select plasmid integrants which restore the e gene. In this work, we demonstrate that DNA inserts more than 7.0 kb in length can be incorporated into a T4 genome lacking the alt gene. In addition, the recombinant T4 not only contains a fusion gene driven by the T4 ipIII promoters, but also packages the fusion protein into the T4 capsid due to targeting by the IPIII portion. This expression-packaging-processing system shows that active IPIII::beta Gal fusion reporter protein is produced and packaged during phage infection.

Bacteriophage T4↗
PubMed1993

Protein folding studies in vivo with a bacteriophage T4 expression-packaging-processing vector that delivers encapsidated fusion proteins into bacteria.

A cloned phage T4 gene which expresses the nonessential capsid scaffold protein IPIII was modified to permit construction and packaging of protein fusions within the capsid. IPIII deletion phage packaged IPIII-beta-galactosidase, IPIII-beta-globin, and IPIII-beta-globin-beta-galactosidase fusion proteins; the latter protein fusion was specifically processed by the T4 gene 21 head morphogenetic proteinase in vivo at a consensus leu(ile)-P1-glu* cleavage site to regenerate beta-galactosidase. Phage inject IPIII-beta-galactosidase protein into bacteria, but less activity is recovered in infections of Escherichia coli dnaK or groEL mutants, suggesting that these host molecular chaperones are required for beta-galactosidase intracellular folding. This expression-packaging-processing (EPP) vector directs protein fusions into capsids for easy detection and purification and permits study of protein delivery and folding in bacteria.

Bacteriophage T4↗