Development and characterization of structural proteins from P23-77, PP7 and Qβ bacteriophages for assembly into virus-like particles

This dissertation explores two complementary approaches for advancing virus-like particle (VLP) technologies from bacteriophages: the expression and characterization of structural proteins from the thermophilic bacteriophage P23-77, and the modification of modular RNA bacteriophage VLPs, PP7 and Qβ, as vaccine platforms. P23-77 infects bacteria that grown at 70 °C. The capsid of the virus is made up of three principal capsid (coat) proteins (VP11, VP16, and VP17) along with several smaller membrane-associated proteins (VP15, VP19, VP20, VP22, and VP23) that may be associated with assembly. Previous studies have assessed the expression of the capsid proteins, but the expression of membrane-associated proteins have never been explored. Additionally, the co-expression of all the structural proteins as well as their potential to assemble to VLPs have never been explored. In the first part of this dissertation, we evaluated the expression and co-expression of these proteins and assessed their potentials to assemble into VLPs. We observed that some of the proteins migrated at sizes greater than their predicted molecular weight and their expression levels depended on the type of purification tags. In silico modeling of individual proteins showed the prevalence of helical and disordered regions. These observations serve as a foundation for future efforts that seek to explore interactions of the structural proteins and the assembly process of P23-77 bacteriophage. The second part of this dissertation focused on modifying coat proteins derived from bacteriophage PP7 and Qβ as platforms for vaccine design. In this part, we assessed the tolerability of bacteriophage Qβ and PP7 coat proteins to insertions of Spytag003 or Spytag peptides; we assessed whether the insertions of these peptides to the coat proteins will affect the ability of the recombinant coat proteins to assemble into VLPs. We observed that the insertion of Spytag003 peptide at either the N- or C-termini of the coat protein of Qβ and at the C-terminus of the coat protein of PP7 affected the solubility of the recombinant proteins. On the contrary, insertion of the shorter Spytag did not affect solubility of PP7 coat proteins. The coat protein of PP7 with Spytag insertions assembled into VLPs displaying Spytag on the surface and could be conjugated with a protein. Immunization of mice with SpyCatcher003 protein conjugated on VLPs elicited better antibodies compared to immunization with the unconjugated protein. Altogether, this dissertation establishes the first experimental foundation for expressing and analyzing P23-77 structural proteins for VLP studies while demonstrating the immunogenic potential of a bio-conjugated protein on PP7 VLPs.