Phage-enzymes, selection

Becker and Hurwitz 94) have found that after infection of E. coli B with T-even bacteriophages a novel 3 -deoxynucleotidase activity appears. They purified the enzyme 2000-fold. In addition to its attack on 3 -deoxymononucleotides, the enzyme selectively removes the 3 -phos-phoryl groups from DNA. It does not attack 3 -ribonucleotides, 3 -phosphoryl groups of RNA, or 5 -phosphate esters. Like bacterial 5 -nucleotidases, this enzyme is markedly activated by Mg2+ and Co2+ and is inhibited by EDTA. The enzyme appears to be a phage-induced enzyme the activity rises early after injection with T-even phages and formation of the enzyme is blocked with chloramphenicol. 

Whenever possible, amodel selection should be optimized before starting selections with a library. In this experiment, a mixture of active and inactive phage-enzymes is used as a model library for one round of selection. The phage mixture is analyzed before and after selection, yielding numbers that serve for the calculation of an enrichment factor (EF) ... 

At least 20 clones resulting from the last round before the plateau is reached should be sequenced for evaluating the diversity after selection. Depending on the complexity of the activity assay, as many clones as possible should also be screened for activity. Monoclonal preparations of phage-enzymes should be assayed first and, if the activity is too low, soluble over expressed enzymes should be produced for reaching higher concentrations. 

As shown in Figure 6.1, the protocol starts with labeling the phages with the substrate. Several approaches have been used for this labeling step [11,13,14]. The active enzymes are then turning over the substrate into product by intraphage catalysis. Finally, the pro duct-labeled phages are selected by classical affinity capture. 

Mukhija S, Erni B, Phage display selection of peptides against enzyme I of the phosphoenolpyruvate-sugar phosphotransferase system, Mol. Microbiol., 25 1159-1166, 1997. 

In order to avoid these limitations, efforts have been made to find ways of coupling substrate turnover directly to a selection process. Two groups have devised a method that involves the attachment of a substrate to a phage-enzyme in a way that allows its intraphage interaction with the enzyme. Phage-displaying active enzymes are able to convert the substrate into product. They can be captured from mixtures with product specific reagents or antibodies. 

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. 

Fig. 5.16. Strategy for the selection of a metallo-P-lactamase (Bla) displayed on phage [69], The phage-enzyme is inactivated by extracting the metallic cofactor and captured with an immobilised penicillin substrate. Addition of the metallic cofactor results in the catalytic elution of the phage-enzyme.

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

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