Selectively-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions

Krebber, Claus; Spada, Stefania; Desplancq, Dominique; Krebber, Anke; Ge, Liming; Plückthun, Andreas

doi:10.1006/jmbi.1997.0981

Cited by 88 publications

(76 citation statements)

References 41 publications

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“…Figure 3A shows the effect of the N1, N1L1, N2, and N1L1N2 regions on wild-type infection. It was obvious that the N1 domain itself has no effect on the wild-type infection, although it has been shown by others that the N1 fragment can have a weak effect at much higher concentrations than those used in these experiments (13). The presence of N2 domain has an inhibitory effect on the wild-type infection, giving an infection that ranges from 20 to 68% of the normal value, while the presence of N1L1 or N1L1N2 almost completely abrogated the infection.…”

Section: Phagementioning

confidence: 75%

“…The exposed coreceptor will facilitate an F-pilus-independent infection through the direct interaction between the N1 domain and the TolA molecule. Therefore, increased efficiency of infection utilizing CaCl 2 has only been observed with N2-deleted g3p mutants, while no apparent effect has been detected by using either N1-deleted g3p mutants or wild-type phages (13,27). The g3p mutant phage constructs, together with wild-type phage, were tested for normal F ϩ TolA ϩ infection in the presence or absence of 50 mM CaCl 2 ( Table 3).…”

Section: Phagementioning

confidence: 99%

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The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

Nilsson

Malmborg

Borrebaeck

2000

J Virol

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The filamentous bacteriophage infects Escherichia coli by interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression.Filamentous bacteriophage is a nonlytic DNA virus that infects Escherichia coli cells harboring an F episome (19,20,26). In order for bacteriophage infection to occur, including the translocation of the single-stranded phage DNA over the cell membrane, the expression of two different receptor systems in the gram-negative host cell is critical. The primary receptor, i.e., the F pilus, mediates the adsorption of the phage to the bacterial cell (6,12). This interaction can take place at a long distance from the cell surface, since the pilus molecule extrudes away from the bacteria. Through the depolymerization of the pilus, the bacteriophage is brought close to the cell surface of the host, where the interaction with the coreceptor takes place (5, 31). The coreceptor, i.e., the TolQRA complex, is localized in the inner membrane and the periplasmic space and is attached to the inside of the outer membrane (9, 16). It was recently shown that it is the C-terminal part of the TolA domain that interacts with the bacteriophage adsorption protein (7,27). Upon binding to the TolA domain, the viral DNA translocates into the host cell by an as-yet-unknown mechanism.Expression of the gene 3 viral adsorption protein, g3p, on the surface of the bacteriophage is essential for normal infection to occur (1). The minor coat protein g3p is located together with g6p in three to five copies at one end of the filamentous phage (10). Mature g3p consists of 406 amino acid residues separated in several distinct regions (2). The N-terminal part can be divided into two different domains, N1 and N2, mediating penetration and adsorption during infection, respectively (1, 30). The C-terminal part is responsible for the interaction with g6p, thereby anchoring the g3p in the membrane of the phage coat (1, 14, 21). Furthermore, the g3p has a crucial role in the assembly of the filamentous phage, since in the absence of g3p, the proper termination of the assembly and the following release of the phage will not take place (24).Two glycine-rich linker regions divid...

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Section: Phagementioning

confidence: 75%

Section: Phagementioning

confidence: 99%

The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

Nilsson

Malmborg

Borrebaeck

2000

J Virol

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“…Phage Infection Experiments-For all experiments, a derivative of the fCKCBS phage was used, which contains the gene for chloramphenicol acetyl transferase and G3P with a modified linker between the N2 and C-terminal domains (15,16). Phage were isolated from E. coli culture medium as described before, resulting in 50-l aliquots (11).…”

Section: Expression Purification and Modification Of The Proteins-mentioning

confidence: 99%

“…2 and supplemental Table 1), trHNCACB and trHN (CO)CACB spectra of a 15 N/ 13 C/ 2 H sample were acquired with a Bruker Avance 800 spectrometer. A 3.6 mM solution of TolA-C was titrated to 0.3 mM [ 15 N]G3P in 14 steps up to a 12-fold excess of TolA-C (1.8 mM TolA-C, 0.15 mM G3P) and monitored by 15 N-TROSY-HSQC spectra with a Bruker Avance 900 spectrometer. Free G3P and bound G3P were in slow exchange on the NMR timescale; therefore, binding was analyzed by the decrease of 1 H 15 N cross-peaks of G3P.…”

Section: Expression Purification and Modification Of The Proteins-mentioning

confidence: 99%

“…To measure amide HX of the complex between G3P and TolA-C, 0.15 mM G3P and 0.60 mM TolA-C were dissolved in D 2 O buffer and the exchange was monitored by collecting 46 15 N-TROSY-HSQC spectra over 91 h (Fig. 3, A 15 N-TROSY-HSQC spectra were acquired successively by a Bruker Avance 900 spectrometer. The first (kinetic) spectrum was obtained during the folding reaction; the second spectrum was measured after the completion of refolding and served as the reference for fully folded G3P.…”

Section: Expression Purification and Modification Of The Proteins-mentioning

confidence: 99%

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Initiation of Phage Infection by Partial Unfolding and Prolyl Isomerization

Hoffmann-Thoms

Weininger

Eckert

et al. 2013

Journal of Biological Chemistry

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Background: Bacteriophage fd is activated for infection by partial unfolding and prolyl isomerization. Results: NMR spectroscopy localized Pro-213-coupled unfolding to regions of the interdomain hinge of the phage gene-3-protein.Conclusion: Pro-213 regulates phage infectivity by a specific long-range effect on the conformational stability of the gene-3-protein.Significance: A proline switch controls the biological function in a remote part of a protein.

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Phage Display Selection and Analysis of Ab‐Binding Epitopes

Enshell-Seijffers

Gershoni

2002

CP in Immunology

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The identification and characterization of B cell epitopes by combinatorial phage display peptide analyses is based on the principle that unique peptides can be affinity-purified from an enormous collection of random peptides. Moreover, once selected, the peptide sequence can be elucidated; filamentous bacteriophages have been genetically engineered to incorporate the DNA template corresponding to the peptide displayed on its surface. This unit begins with a discussion of some of the factors that distinguish available libraries. Protocols are then provided for affinity selection of antibody-specific phages, determination of phage titer, confirmation and amplification of positive phages, phage characterization, and construction of custom-tailored phages. The selection protocol in this unit is specific and designed for libraries that are used in the authors' laboratory and are based on the fth1 or fd-tet derived vectors. However, information is included for adapting these protocols to the specific requirements of other phage display libraries.

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Selectively-infective phage (SIP): a mechanistic dissection of a novel in vivo selection for protein-ligand interactions

Cited by 88 publications

References 41 publications

The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

The Phage Infection Process: a Functional Role for the Distal Linker Region of Bacteriophage Protein 3

Initiation of Phage Infection by Partial Unfolding and Prolyl Isomerization

Phage Display Selection and Analysis of Ab‐Binding Epitopes

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