Biology subjects

Tay, N. E. S.

Publications and source records attributed to Tay, N. E. S..

3 recordsLinked to original sources

bioRxiv2024

Protein Editing using a Concerted Transposition Reaction

Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes in vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an in vitro strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this protein transposition reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multi-subunit chromatin remodeling complex, ACF, where we also show protein transposition can occur in situ within the cell nucleus. By carrying out a molecular cut and paste on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.

biochemistry↗

bioRxiv2023

Cell surface sculpting using logic-gated protein actuators

Cell differentiation and tissue specialization lead to unique cellular surface landscapes and exacerbated or loss of expression patterns can result in further heterogenicity distinctive of pathological phenotypes1-3. Immunotherapies and emerging protein therapeutics seek to exploit such differences by engaging cell populations selectively based on their surface markers. Since a single surface antigen rarely defines a specific cell type4,5, the development of programmable molecular systems that integrate multiple cell surface features to convert on-target inputs to user-defined outputs is highly desirable. Here, we describe an autonomous decision-making protein device driven by proximity-gated protein trans-splicing that allows local generation of an active protein from two otherwise inactive fragments. We show that this protein actuator platform can perform various Boolean logic operations on cell surfaces, allowing highly selective recruitment of enzymatic and cytotoxic activities to specific cells within mixed populations. Due to its intrinsic modularity and tunability, this technology is expected to be compatible with different types of inputs, targeting modalities and functional outputs, and as such will have broad application in the synthetic biology and biotechnology areas.

synthetic biology↗

bioRxiv2020

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.

microbiology↗