Biology subjects

Koch, N. G.

Publications and source records attributed to Koch, N. G..

2 recordsLinked to original sources

bioRxiv2024

Amber Codon Mutational Scanning and Bioorthogonal PEGylation for Mapping Antibody Binding Sites on Human Arginase-1

Epitope mapping is crucial for understanding immunological responses to protein therapeutics. Here, we combined genetic code expansion and bacterial surface display to incorporate S-allylcysteine (SAC) into human arginase-1 (hArg1) via Methanococcoides burtonii pyrrolysyl-tRNA synthetase. Using an amber codon deep mutational scanning and sequencing workflow, we mapped SAC incorporation efficiency across the hArg1 sequence, providing insights into structural and sequence dependencies of non-canonical amino acid incorporation. We used mutually bioorthogonal allyl/tetrazine and azide/DBCO chemistries to achieve site-specific PEGylation and fluorescent labeling of hArg1, revealing insights into SAC side chain reactivity and solvent accessibility of residues in hArg1. This system was further applied to determine the binding epitope of a monoclonal antibody on the surface of hArg1, providing high-resolution data on the impact of PEGylation residue position on antibody binding. Our method produces high dimensional data of non-canonical amino acid incorporation efficiency, site-specific functionalization enabled by mutually bioorthogonal chemistries, and epitope mapping of therapeutic proteins.

bioengineering↗

bioRxiv2023

"Cold" Orthogonal Translation: Psychrophilic Pyrrolysyl-tRNA Synthetase as Efficient Tool for Expanding the Genetic Code

Using orthogonal translation systems (OTSs) is one of the most efficient strategies for producing unnatural proteins through the incorporation of non-canonical amino acids (ncAAs) into the genetic code. Traditionally, efforts to expand substrate specificity start with a (hyper-)stable enzyme capable of withstanding the structural changes induced by necessary mutations. In contrast, we propose a radically different approach for the PylRS system: by starting with enzymes that evolved to cope with instability in order to adapt to cold conditions, potentially offering greater resilience to mutational changes. By finding and further engineering a psychrophilic ("cold") OTS from Methanococcoides burtonii, we developed an alternative to the widely used mesophilic and thermophilic systems. This novel OTS demonstrated exceptional in vivo efficiency for a broad range of substrates, even at very low ncAA concentrations and low cultivation temperatures. The general versatility of the PylRS system across a wide range of applicable host organisms suggests that the Cold-OTS has the potential to also improve protein yields in these hosts and help to drive the transformation of the expanded genetic code from an academic pursuit into a high-value, chemistry-driven biotechnology.

synthetic biology↗