Abstract
The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.
Highlights
Following its discovery by George Smith in the early 1980’s, phage display technologies have been built predominantly from DNA phage platforms, that of M13 [1,2,3,4,5]
In this report we present a framework of peptide display and affinity maturation using Qb phage and the integrin receptor of Foot-and-MouthDisease-Virus (FMDV) as a proof-of-concept for acquiring binders to a highly infectious agent with many different serotypes
We show that 3 different gene fusions with A1 placed in front of the natural opal and ochre stop codons (TGA and TAA), produced phage plaques in bacterial lawns (Fig. 2)
Summary
Following its discovery by George Smith in the early 1980’s, phage display technologies have been built predominantly from DNA phage platforms, that of M13 [1,2,3,4,5]. M13 is DNA-filamentous bacteriophage with a genome size of 6.4 kb [6] and have very low mutation rates that limit their use in in vitro evolution processes. RNA-based replication systems possess attractive features, including high mutation rates, high population size and short replication times, that can be exploited for rapid in vitro evolution [7]. Efforts to generate recombinant RNA had limited success due to limitations in technology and RNA instability. Recent advancements have led to the generation and cloning of Qb cDNA into several stable plasmids that are able to liberate phage upon bacterial transformation [8]. The cDNA of Qb coliphage RNA has become amenable for use in displaying random peptide libraries in vitro followed by in vivo translation and phage production
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