Phage-enzymes

A second enzyme on the pathway to dTTP that is subject to allosteric control is deoxycytidylate deaminase, which supplies dUMP for thymidylate synthesis. The enzyme in mammalian cells, yeast, and bacteriophage T2-infected E. coli. is allosterically activated by dCTP (hydroxymethyl dCTP for the phage enzyme) and inhibited by dTTP. 

Here are two examples of peptide linkers that we have successfully used with phage-enzymes ... 

A solution of phage-enzyme can be used like an enzyme solution for measuring kinetic parameters such as kcat and KM. We usually observed that phage-displayed enzymes behave essentially like free enzymes in solution, although interference is always possible, especially with multiple display. Note that the kcat is the turnover rate of the phage and not of the enzyme, because the level of display is generally just an evaluation (see below). 

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. 

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. 

Tetra, octa, and dodecasaccharides have been prepared from Salmonella typhimurium lipopolysaccharide by specific degradation of the O chain with phage enzyme. These oligosaccharides contain 1,2, or 3 chemical repeating units (Svenson and Lindberg, 1981). 

Phage P22 apparently requires the presence of the dideoxyhexosyl group, since an abequose-deficient LPS was not cleaved by the phage enzyme (33). On the other hand, two other phages, 28B and 36, with endoglycosidase activity against the L-Rha (al->-3)D-Gal linkage were able to cleave the abequose-deficient LPS (36). 

The phage enzyme-bacterial PS substrate interaction resulted in the release of oligosaccharides. When crude preparations,... 

McCafferty J, Jackson RH, Chiswell DJ, Phage-enzymes expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage, Protein Eng., 4 955-961, 1992. 

The concentration of displayed enzyme is evaluated by measuring the activity of the phage-enzyme solution with the assumption that the displayed enzyme has the same activity as the free enzyme in solution. In our experience, this is generally the case. 

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. 

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