The biology of filamentous phage infection - implications for display technology
Abstract: Phage display technology is a biotechnological tool that can be used to obtain molecules/reagents able to discriminate between target molecules. Reagents with such features can be used in diverse settings like biological chemistry, diagnostics and in therapeutic applications. Phage display technology is based on the use of recombinant DNA technology, bacteria and a special kind of bacterial virus, the filamentous phage. However, the chance of obtaining high binding strength (affinity) reagents using conventional phage display is limited by e. g. the selection procedure. Therefore, protocols that increase the likelihood of recovering high affinity binding proteins from a pool of similar molecules, a so called library, are of interest. One approach is to modify the selection procedure of phage display, which has generated a special application called selective infection. Another approach is to study the infection process of filamentous phage, in hope of revealing mechanistic events suitable to manipulation. In this thesis, which is based on four original papers, selective infection is evaluated and the infection mechanism of filamentous phage is assessed in conjunction to implications for phage display technology. By analysing the prerequisite for successful selective infection, we found a correlation between the affinity of the interacting pairs and infection efficiency and that a phage format allowing multiple display of binding proteins was superior to one that does not allow such multiple display. Thus, selective infection has characteristics that make it suitable for retrieving mainly high affinity binding proteins from phage display libraries. The infection mechanism of filamentous phage was dissected by analysing the molecular interactions between phage coat protein pIII and the bacterial co-receptor protein of phage infection, TolA. The binding affinities between these proteins and their different domains were characterised and novel interactions were detected, allowing for a refined hypothetical model of the infection mechanism of filamentous phage. Furthermore, TolA mutants were created for the analysis of its phage receptor and outer membrane integrity functions. It was found that these two TolA functions could be segregated, thus emphasising the mechanistic differences between the two functions of TolA. Finally, the changes in gene expression of phage infected Escherichia coli, was monitored by global transcription analysis and it was demonstrated that several host genes were co-ordinately affected. In conclusion, these studies have provided a basis for the development of phage display technology, as well as insights into several different aspects of the infection process of filamentous phage.
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