Browsing by Author "Waffo, Alain B."

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    Mapping the Major Epitope(s) of the Glycoprotein of Ebolavirus with QB Phage Display System
    (2023-02) Edwards, Andrew; Waffo, Alain B.; Elmendorf, Jeffrey S.; Quilliam, Lawrence A.
    The main goal of the research was to produce and analyze Qβ phage virions containing various segments of the EBOV glycoprotein fused to its A1 the readthrough minor coat protein. The recombinant phages would then be utilized to analyze their antigenicity and to map the major epitope(s) of the glycoprotein determinants. This research study represents a proof of concept and will serve as a guide as to how to produce recombinant phages bearing large antigenic peptide segments of a viral protein and initiate its analysis. To prepare the recombinant plasmids, separately, the gene segments of the glycoprotein determinants were designed with overlapping fragments, fused with the A1 gene and amplified by PCR. The obtained gene fragments were separately purified, digested and ligated with a modified phage display plasmid vector pBR322 containing a full copy of cDNA genome of the Qβ phage. The resulting ligation was used to transform E. coli DH5α competent cells. Colonies were picked, grown overnight, plasmids purified, analyzed using restriction enzymes, and confirmed via Sanger sequencing. Positively sequenced plasmid clones were used to retransform E. coli HB101 for phages production. The phage titer was 105 pfu/ml, lower than that typically achieved with wildtype phages. The recovery and resuspension of the phages from PEG/NaCl was further scaled to a titer of 1012 pfu/ml, followed by precipitation, and RNA isolation. The recombinant RNA was used to obtain cDNA for PCR amplification. The amplified cDNA was analyzed by agarose gel electrophoresis and sequenced to confirm the presence, position, and orientation of the glycoprotein segment within the recombinant phages. We were able to successfully produce recombinant phages harboring gene segments of the Ebolavirus glycoprotein fused to the Qβ phages A1 coat protein. All the phages with the glycoprotein determinants will be analyzed for their antigenicity using specific glycoprotein antibody once they are available. Additionally, our panning methodology will be used to determine the segment(s) containing the major epitopes. The segment will be confirmed using blotting and agarose double diffusion technique. This work can be extended to identify antibody epitopes in other RNA viruses and as a point of care for major infectious diseases.
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    Uniqueness of RNA Coliphage Qβ Display System in Directed Evolutionary Biotechnology
    (MDPI, 2021-04) Nchinda, Godwin W.; Al-Atoom, Nadia; Coats, Mamie T.; Cameron, Jacqueline M.; Waffo, Alain B.; Biochemistry and Molecular Biology, School of Medicine
    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.