Phage bound

Add 1 5 mL of blocked streptavidin Dynabeads to the phage bound to S-S biotinylated antigen... 

A possible limitation of the selection protocols described above arises from the intramolecular nature of the process. A single catalytic event, transforming the phage-bound substrate into product during the time required to complete the experiment is sufficient to lead to selection of the phage-enzyme. Consequently, poorly active enzymes are likely to be selected. 

Wash the cells as before and elute all phage bound to the cells by adding 100 pi of freshly made 100 mM triethylamine and incubating at room temperature for 10 min. 

Cro, by contrast, acts purely as a repressor. When it is bound to its high-affinity site at OR3, it prevents repressor synthesis by obstructing the access of polymerase to the left-hand promoter. In the absence of repressor, RNA polymerase can bind to the Cro promoter, and Cro can be synthesized along with the early phage genes to its right. 

How do the mutations identified by phage display improve binding specificity There is as yet no direct stmctural information on the phage-selected inhibitors however they can be modeled using data from the crystal structures of other Kunitz domains bound to serine proteinases. These studies lead to the conclusion that the mutations identified by phage display improve binding specificity by maximizing complementarity between the... 

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,...

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