PubMed · 7272335
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L Vassilev, G Russev. 1981. Ion activity decrease in the presence of sucrose.. https://doi.org/10.1016/0304-4165(81)90229-4
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Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.
Tn10 transposes nonreplicatively. Staged in vitro reactions demonstrate that a Tn10 synaptic complex can become committed to a particular target DNA molecule via a noncovalent interaction in the absence of strand transfer. Commitment occurs only after double-strand cleavage at both transposon ends (in "double-end break" [DEB] complexes). Stable noncovalent DEB-target DNA cocomplexes can be detected, but no cocomplexes occur with synaptic complexes containing uncleaved ends. Preincubation of DEB complexes with target DNA accelerates the rate of strand transfer. Postcleavage target capture is remarkable for Tn10; Mu and Tn7 select a target site prior to cleavage. Promiscuous target selection may favor evolution of IS-based composite elements while being suicidal for other types of transposons.
Surface plasmon resonance technology was used to study the binding of activated factor VII to tissue factor, in the presence of divalent metal ions. The binding was calcium-dependent, with the optimum calcium concentration at 5 mM. Only very minimal binding was observed in the absence of added calcium ions and presence of 10 mM EGTA. The effect of the calcium concentration on the apparent dissociation constants for the TF/VIIa complex formation was also investigated. In the absence of calcium ions but in the presence of either magnesium or zinc ions, no significant binding of activated factor VII to tissue factor was detected. However some binding was observed with manganese ions, indicating that manganese ions could partially replace calcium ions to support the formation of the TF/VIIa complex. These results are consistent with studies of the effect of divalent metal ions on the amidolytic activity of the TF/VIIa complex.