Phages wild-type

Andrew Braisted and J.A. Wells prepared phage containing Z domain helices 1 and 2 and restored Fc binding of this 38 residue minidomain in three iterative stages (see Figure 17.10). The truncated peptide was first randomized at four hydrophobic residues which contact helix 3 in the complete Z domain. The consensus sequence from this library maintained the wild-type residues lie 17 and Leu 23 while the hydrophobic residues Leu 20 and... 

Figure 17.10 Construction of a two helix truncated Z domain, (a) Diagram of the three-helix bundle Z domain of protein A (blue) bound to the Fc fragment of IgG (green). The third helix stabilizes the two Fc-binding helices, (b) Three phage-display libraries of the truncated Z-domaln peptide were selected for binding to the Fc. First, four residues at the former helix 3 interface ("exoface") were sorted the consensus sequence from this library was used as the template for an "intrafece" library, in which residues between helices 1 and 2 were randomized. The most active sequence from this library was used as a template for five libraries in which residues on the Fc-binding face ("interface") were randomized. Colored residues were randomized blue residues were conserved as the wild-type amino acid while yellow residues reached a nonwild-type consensus, [(b) Adapted from A.C. Braisted and J.A. Wells,...

A. nidulans with selected phages of classes A, B, C, D and E resulted in increased polygalacturonase activity in comparison with the wild type strain. The culture media of pga transformants were further analysed by Western blotting with polyclonal antibodies raised against PGI and in all samples examined cross-reactive bands with molecular masses similar to that found for PGI or PGII were detected. 

Exit of the virus from the cell occurs as a result of cell lysis. The phage codes for a lytic enzyme, the T4 lysozyme, which causes an attack on the peptidoglycan of the host cell. The burst size of the virus (the average number of phage particies per cell) depends upon how rapidly lysis occurs. If lysis occurs early, then a smaller burst size occurs, whereas slower lysis leads to a higher burst size. The wild type phage exhibits the phenomenon of lysis inhibition, and therefore has a large burst size, but rapid lysis mutants, in which lysis occurs early, show smaller burst sizes. 

The level of display is the average number of enzymes displayed per phage particle. For a wild-type enzyme whose activity is not affected by the phage environment, the level of display is evaluated by dividing the kC3t of the phage by the /ccat of the free enzyme. Otherwise, it can be evaluated by Western blot or, when possible, by active-site labeling. 

Gillin, F. D., and Nossal, N. G. (1976). Control of mutation frequency by bacteriophage T4 DNA polymerase II. Accuracy of nucleotide selection by the L88 mutator, CB120 antimutator, and wild type phage T4 DNA polymerases. J. Biol. Chem. 251, 5225-5232. 

FIGURE 16.4 Schematic representation of phage (b) and phagemid (c) display of polypeptides fused to pill and pVIII coat proteins, compared to wild-type phage (a). 

Competition at the Outer Membrane. Ferrichrome Receptor. Escherichia coli. It was easily demonstrated that tonA and tonB mutants were also resistant to albomycin (I, 35). The first experiments to test competition were performed by simply adding ferrichrome to wild type cells plus phage in a plate assay. Later an adsorption assay was used to... 

In contrast, in phage T4 lysozyme a thermolabile mutant was found in which one single amino acid was exchanged Thr- Ile 157, and the thermal melting point dropped from 42°C for the wild-type enzyme to 31 °C for the mutant. Systematic exchange of the amino acid in position 157 by site-directed mutagenesis led to the... 

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