Protein Engineering with Biosynthesized Libraries from Bordetella bronchiseptica Bacteriophage

Abstract

Phage display offers a powerful approach to engineer protein affinity. A naturally occurring analog to phage display, the Bordetella bronchiseptica bacteriophage (BP) employs a highly variable protein termed the major tropism determinant (Mtd) to recognize its dynamic host. Propagation of BP provides a self-made phage library (SMPL) with vast numbers of phage particles, each displaying a single Mtd variant. We report applying the diversity of the BP-SMPL to access a tyrosine-rich library of Mtd variants. Expression of the SMPL-engineered Mtd variant as a GST-bound fusion protein demonstrated specific binding to the target T4 lysozyme with dissociation constants in the sub-micromolar range. The results guide future experiments with SMPLs applied to protein engineering.

Prokaryotic reverse transcriptases: from retroelements to specialized defense systems

“…However, only one preliminary study has been able to demonstrate the ability of the phage BPP-1 DGR to create variants of tropism proteins likely to bind the T4 lysozyme (Table 1 ; Yuan et al . 2013 ). The exploration of novel DGRs with advantageous properties, such as thermostability (Handa, Shaw and Ghosh 2019 ), could provide promising new tools.…”

Modern methods for laboratory diversification of biomolecules

“…The mutagenized cDNA is integrated into a stable genomic locus, at which point the biomolecule variant can be assessed by functional selection or screen. Related mutagenesis systems are also known occur naturally, such as in the Bordetella bronchiseptica bacteriophage retroelement, which is comprised of an error-prone reverse transcriptase that selectively mutates codons of a key surface protein that determines host specificity [40]. This natural phage-display system has been coopted for use in the laboratory to evolve high-affinity T4 lysozyme binders, relying exclusively on the natural diversity generated by the retroelement [40].…”

Evolving a Peptide: Library Platforms and Diversification Strategies

“…In eukaryotes, this was overcome by the development of orthogonal in vivo DNA replication apparatus, which in essence utilizes plasmid-polymerase pairs, limiting mutagenesis to a cytoplasm-only event [206]. Related phenomena are also known to occur in nature (e.g., the Bordetella bronchiseptica bacteriophage error-prone retroelement, which selectively introduces mutations into the gene encoding the major tropism determinant (Mtd) protein on the phage tail fibers [207]) and can be exploited for creating libraries [208].…”