Substrate phage

Vectors constructed with such proteolytic linkers can thus be released from their target during panning with a mild buffer containing the appropriate protease, without having to guess at optimal elution conditions (see Ref. 85 thrombin cleavage for elution). 

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In addition to selecting peptides for metal substrates, phage display methods successfully identified peptides capable of forming sihea nanospheres. Recently, many groups have isolated proteins and peptides responsible for the in vivo prodnetion of sihea. Stone and coworkers have isolated additional novel peptides nsing phage display that are capable... 

Chen, I., Choi, Y. A. and Ting, A. Y. (2007). Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins. J. Am. Chem. Soc. 129, 6619-25. 

More recently, Janda has described the production of a galactopyranosidase antibody in response to hapten [96]. This was designed to accommodate several features of the transition state for glycoside hydrolysis notably a flattened half-chair conformation and substantial sp2 character at the anomeric position. Some 100 clones were isolated in response to immunization with [96] and used to generate a cDNA library for display on the surface of phage (Appendix entry 7.3) (Janda et al., 1997). Rather than proceed to the normal screening for turnover, Janda then created a suicide substrate system to trap the catalytic species. 

Figure 21 Hapten 35 and fluorogenic substrates 36-38 used to screen phage displayed Fabs with an aldolase activity.

Figure 4.6 The phage display scheme for the production of never-born proteins. Proline-arginine-glycine (PRG), is a substrate for thrombin, and TAG is the antibody target. (Modified from Chiarabelli et al, in press.)...

Phage display techniques can be used in any field where molecular biological approaches find application. It can be successfully used for epitope mapping, vaccine development, identification of proteinkinase substrates and nonpeptid ligands, selection of functional interactions of hormones, enzymes, enzyme inhibitors, mapping... 

Students may characterize A phage or adenovirus 2 DNA. Both are standard substrates for assays of restriction endonucleases. 

Fig. 6.1. Summary of methods for selecting phage-displayed enzymes on die basis of catalytic activity (S substrate P product SS suicide substrate Bt biotin SV streptavidin TSA transition state analogue).

The following protocol is described for the selection of phage-displayed serine / -lactamase with a biotinylated penam-sulfone [5] suicide substrate. For other activities, the concentrations of substrate and suicide substrate and the times of reaction should probably be adjusted. 

In a final volume of 1 mL phosphate buffer (50 mM, pH 7.0), mix 1012 phages with 10-5 M of substrate and incubate 10 min at room temperature to block all the active sites that are inactive. 

Working with an immobilized substrate, however, can be a source of problems that are difficult to identify. Because of the slow diffusion of the phages, the time of reaction should be greatly increased. Control of some parameters, such as interference by the support or the density of immobilization, is difficult. Moreover, substrate recognition by the enzyme can be significantly impaired. 

In this strategy, phages are affinity-captured on immobilized substrate under conditions in which the enzyme is inactive, for example, in the absence of an essential metal ion or a cofactor. Active phage-enzymes are then eluted by triggering catalysis by addition of the metal ion or cofactor, taking advantage of the lower affinity for the product than for the substrate. 

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