Abstract
BackgroundThe promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.ResultsFluorescence activated cell sorting (FACS) of 1.5 × 107 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L.ConclusionsFACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.
Highlights
The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains
In this report we demonstrate that flow cytometric sorting of small samples of highly diverse yeast-displayed Type III human fibronectin domain (Fn3) libraries is an effective method for de novo isolation and affinity maturation of virion-binding protein domains
Flow cytometric sorting was performed on only 1.5 × 107 clones derived from a Fn3 library with a clonal diversity exceeding 4 × 109, yielding two unique Zika virion binders that were subsequently subjected to affinity maturation to obtain variants with single-digit nanomolar virion binding affinity
Summary
The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Construction and FACS of sitedirected binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zikabinding affinities These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L. It is possible that extracellular segments of viral envelope proteins can be expressed as soluble proteins/peptides for binder discovery, but these “out-of-context” proteins/peptides might not faithfully recapitulate the secondary/tertiary structure present on the viral surface These considerations make isolation and engineering of virion-binding domains by screening against intact virions a more desirable approach to obtaining pools of virion-binding domains than multiplex screening against collections of recombinantly expressed virion surface proteins/peptides. The most recent body of reported work involving de novo isolation of surface-displayed binder libraries
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