scholarly journals Uniqueness of RNA Coliphage Qβ Display System in Directed Evolutionary Biotechnology

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 568
Author(s):  
Godwin W. Nchinda ◽  
Nadia Al-Atoom ◽  
Mamie T. Coats ◽  
Jacqueline M. Cameron ◽  
Alain Bopda Waffo

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.

1998 ◽  
Vol 80 (09) ◽  
pp. 354-362 ◽  
Author(s):  
Begoña Arza ◽  
Jordi Félez

IntroducationThe phage display technology represents a powerful tool for protein and drug design because vast numbers of amino acid sequences can be rapidly explored. This review describes the origins of phage libraries, their evolution and more recent advances, including examples in the area of thrombosis and haemostasis, where the phage display approach has just begun to be used with great success.Phage display uses filamentous phages (E. coli specific phages with a filamentous shape that contain a single stranded closed circular molecule of DNA), such as M13 or fd, as vehicles for displaying foreign peptides or proteins on their surface. This is carried out by fusing the coding sequence (DNA) for the peptide or protein to the amino (N)-terminus of either full-length phage minor coat protein III (cpIII), or to phage major coat protein VIII (cpVIII), to carboxy (C)-terminal domain of cpIII or, more recently, by fusion to the C-terminus of full-length phage minor coat protein VI (cpVI) (Fig. 1). Expression of the fusion protein and its subsequent incorporation into the mature phage particle results in the foreign peptide or protein being presented on the phage surface. Thus, the linkage of each peptide or protein to its encoding genetic material [contained as part of the single-stranded viral DNA (1, 2)] represents a great advantage over conventional cloning methods.The phage display approach was first used by Smith in 1985 (3), who expressed a library of peptide sequences at the N-terminus of cpIII (3-5). This insertion allowed phage assembly and display of the peptide on the phage surface, without affecting the phage infectivity significantly. The linkage of genotype and phenotype in the phage library allowed facile isolation of clones of specific interest from pools of millions of clones by successive rounds of phage affinity selection on surfaces coated with a ligand (panning) followed by phage amplification by infecting male E. coli (Fig. 2).The foreign peptides or proteins were displayed, initially, on every copy of the coat protein, but only short peptides can be displayed in this way without altering the phage infectivity. Two systems have been developed to solve this problem. One incorporates a second native gene III or VIII in the phage genome giving a mixture of native and recombinant coat protein incorporation on the phage. The second system provides the recombinant cpIII, cpVIII or cpVI gene on a phagemid (a plasmid containing the origin of replication of filamentous phage). Phagemids can be packaged into phage particles by superinfection with a helper phage. The fusion protein is incorporated onto the surface coat, along with copies of the native coat protein encoded by the helper phage. The result is a mixture of wild-type helper phage and recombinant phagemid particles, but due to a defective origin of replication the helper phage is poorly packaged to provide minimal competition with the phagemids (6). Phages that display both native coat protein and fusion protein are infective. Therefore, the display systems can be either multivalent or monovalent. Multivalent systems make use of gene III phage constructs or gene VIII phage or phagemid constructs and give a high number of foreign domains displayed on their surface (7). Monovalent systems utilize gene III or gene VI phagemid constructs, which have a low number of foreign domains displayed on their surface, usually a single copy. Consequently, monovalent systems distinguish between low affinity and high affinity clones in panning assays (1, 8).


2020 ◽  
Vol 26 (42) ◽  
pp. 7672-7693 ◽  
Author(s):  
Bifang He ◽  
Anthony Mackitz Dzisoo ◽  
Ratmir Derda ◽  
Jian Huang

Background: Phage display is a powerful and versatile technology for the identification of peptide ligands binding to multiple targets, which has been successfully employed in various fields, such as diagnostics and therapeutics, drug-delivery and material science. The integration of next generation sequencing technology with phage display makes this methodology more productive. With the widespread use of this technique and the fast accumulation of phage display data, databases for these data and computational methods have become an indispensable part in this community. This review aims to summarize and discuss recent progress in the development and application of computational methods in the field of phage display. Methods: We undertook a comprehensive search of bioinformatics resources and computational methods for phage display data via Google Scholar and PubMed. The methods and tools were further divided into different categories according to their uses. Results: We described seven special or relevant databases for phage display data, which provided an evidence-based source for phage display researchers to clean their biopanning results. These databases can identify and report possible target-unrelated peptides (TUPs), thereby excluding false-positive data from peptides obtained from phage display screening experiments. More than 20 computational methods for analyzing biopanning data were also reviewed. These methods were classified into computational methods for reporting TUPs, for predicting epitopes and for analyzing next generation phage display data. Conclusion: The current bioinformatics archives, methods and tools reviewed here have benefitted the biopanning community. To develop better or new computational tools, some promising directions are also discussed.


2001 ◽  
Vol 4 (7) ◽  
pp. 553-572 ◽  
Author(s):  
D. Rodi ◽  
G. Agoston ◽  
R. Manon ◽  
R. Lapcevich ◽  
S. Green ◽  
...  

PLoS ONE ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. e53264 ◽  
Author(s):  
Jinhua Dong ◽  
Takahiro Otsuki ◽  
Tatsuya Kato ◽  
Tetsuya Kohsaka ◽  
Kazunori Ike ◽  
...  

2014 ◽  
Vol 33 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Sara Mohammadzadeh ◽  
Masoumeh Rajabibazl ◽  
Mehdi Fourozandeh ◽  
Mohammad Javad Rasaee ◽  
Fatemeh Rahbarizadeh ◽  
...  

2012 ◽  
Vol 56 (9) ◽  
pp. 4569-4582 ◽  
Author(s):  
Johnny X. Huang ◽  
Sharon L. Bishop-Hurley ◽  
Matthew A. Cooper

ABSTRACTThe vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.


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