Uniqueness of RNA Coliphage Qβ Display System in Directed Evolutionary Biotechnology

Abstract

Phage display technology involves the surface genetic engineering of phages to expose desirable proteins or peptides whose gene sequences are packaged within phage genomes, thereby rendering direct linkage between genotype with phenotype feasible. This has resulted in phage display systems becoming invaluable components of directed evolutionary biotechnology. The M13 is a DNA phage display system which dominates this technology and usually involves selected proteins or peptides being displayed through surface engineering of its minor coat proteins. The displayed protein or peptide’s functionality is often highly reduced due to harsh treatment of M13 variants. Recently, we developed a novel phage display system using the coliphage Qβ as a nano-biotechnology platform. The coliphage Qβ is an RNA phage belonging to the family of Leviviridae, a long investigated virus. Qβ phages exist as a quasispecies and possess features making them comparatively more suitable and unique for directed evolutionary biotechnology. As a quasispecies, Qβ benefits from the promiscuity of its RNA dependent RNA polymerase replicase, which lacks proofreading activity, and thereby permits rapid variant generation, mutation, and adaptation. The minor coat protein of Qβ is the readthrough protein, A1. It shares the same initiation codon with the major coat protein and is produced each time the ribosome translates the UGA stop codon of the major coat protein with the of misincorporation of tryptophan. This misincorporation occurs at a low level (1/15). Per convention and definition, A1 is the target for display technology, as this minor coat protein does not play a role in initiating the life cycle of Qβ phage like the pIII of M13. The maturation protein A2 of Qβ initiates the life cycle by binding to the pilus of the F+ host bacteria. The extension of the A1 protein with a foreign peptide probe recognizes and binds to the target freely, while the A2 initiates the infection. This avoids any disturbance of the complex and the necessity for acidic elution and neutralization prior to infection. The combined use of both the A1 and A2 proteins of Qβ in this display system allows for novel bio-panning, in vitro maturation, and evolution. Additionally, methods for large library size construction have been improved with our directed evolutionary phage display system. This novel phage display technology allows 12 copies of a specific desired peptide to be displayed on the exterior surface of Qβ in uniform distribution at the corners of the phage icosahedron. Through the recently optimized subtractive bio-panning strategy, fusion probes containing up to 80 amino acids altogether with linkers, can be displayed for target selection. Thus, combined uniqueness of its genome, structure, and proteins make the Qβ phage a desirable suitable innovation applicable in affinity maturation and directed evolutionary biotechnology. The evolutionary adaptability of the Qβ phage display strategy is still in its infancy. However, it has the potential to evolve functional domains of the desirable proteins, glycoproteins, and lipoproteins, rendering them superior to their natural counterparts.

Engineered Phage-Based Cancer Vaccines: Current Advances and Future Directions

“…It infects host cells using the F pilus of E. coli, similar to the MS2 phage, and the A2 protein can cause lysis by inhibiting cell wall synthesis [ 140 ]. Qβ phages can also express the A1 protein, which displays antigens through its β-hairpin in the read-through domain consisting of 196 amino acids [ 141 , 142 ]. The display of tumor-specific antigens on the surface of engineered Qβ phages can be used for inducing an immune response against cancer cells.…”

Viral Nanoparticles‐Mediated Delivery of Therapeutic Cargo

“…The stability of Qβ‐VLPs is due to multiple inter‐monomer disulfide bonds (there are four disulfide bonds between each subunit). [ 107 ] The surface charge of Qβ is positive; therefore, the half‐life of the particles is longer than 3 h. However, the half‐life of particles with a negative surface charge is less than 15 min. Chemical modifications can alter the surface charge.…”