Selection of phage-enzymes

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. 

Directed enzyme evolution using the selection of phage-clisplayed enzymes is a powerful tool that represents an attractive alternative to the popular high-throughput screening technologies. 

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... 

The objective of the experiment is to evaluate the action of restriction enzymes on bacterial plasmids, A phage DNA, or viral DNA. The DNA will be incubated under the appropriate conditions with selected restriction enzymes. The reaction mixtures will be subjected to agarose gel electrophoresis in order to determine the number and molecular size of the restriction fragments. 

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) ... 

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