Self-assembling protein scaffolds known as virus-like particles (VLPs) are promising delivery vehicles for diagnostic and therapeutic applications. VLPs are easily produced in a bacterial expression system, are biocompatible, and have defined chemical handles that can attach targeting ligands. However, there are some key challenges to be solved. In particular, VLPs are highly stable and must come apart upon entry to the target cell. Moreover, it is difficult to attain high levels of expression for VLPs that are capable of modification with reliable bio-orthogonal conjugation chemistries. Here, we engineered VLPs based on the MS2 bacteriophage by combining comprehensive codon mutagenesis, one-pot selections, and high-throughput sequencing to create protein fitness maps in a strategy we termed Systematic Mutagenesis and Particle Selection (SyMAPS). Using SyMAPS, we identified several mutations that confer a desired acid instability, as well as residues in the protein that were amenable to multiple mutations. We were intrigued by these mutable residues because several of them were calculated to be as malleable as the current low yield MS2 non-canonical (ncAA) mutant used for exterior modification in drug delivery applications. Here, we adapted the SyMAPS screen to quantitatively asses the ncAA incorporation tolerance for every residue in MS2.

Systematic engineering of virus-like particles to identify self-assembly rules for non-canonical amino acid mutants

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.