The Phage-enzyme

The pill protein is expressed as a precursor having an amino-terminal signal peptide necessary for addressing the protein through the periplasm of E. coli. The signal peptide is removed by a specific protease after secretion, and the pill ends up anchored in the bacterial inner membrane. Its assembly in the phage particle is concomitant with phage extrusion. 

The following protocols have been used for phage vectors, although most of them can be directly applied to phagemids. 

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

Here are two practical protocols for phage production starting from a stock culture of infected bacteria (stored in 40% glycerol at —80 °C)  

Carefully discard the supernatant. Centrifuge again at 10 000 rpm for 1 min and remove the residual liquid. 

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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. 

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,... 

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. 

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.

Although a stem—loop and a 3 U-tract constitute the structural backbone for T7 terminators, the efficiency of termination is influenced by multiple factors such as sequence, stability, and length of the stem—loop, length and context of the U-tracts, promoter type, and distance between the promoter and termination site. Sequences upstream from the canonical stem—loop structure also exert marked effects on the position and efficiency of termination (130). Stem-loop structures that lack a 3 U-tract are not functional as a terminator for the phage enzyme. In contrast, U-tracts having certain 5 sequences that lack an apparent stem-loop structure can function as terminators (131). 

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. 

Figure 5.26 Integration of lambda DNA into the host. Integration always occurs at a specific site on the host DNA, involving a specific attachment site (att) on the phage. Some of the host genes near the attachment site are given. A sitespecific enzyme (integrase) is involved, and specific pairing of the complementary ends results in integration of phage DNA.

Load one sample into each of the sample wells in the following manner. Mix each 20 fiL of reaction mixture from the restriction enzyme with 10 fxL of gel-loading buffer and apply one 10 nL sample to each well. Put 10 fiL of roRI standard digest of X phage DNA into one sample well. 

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