Cell Penetration Profiling using the Chloroalkane Penetration Assay for Peptides, Proteins and Nucleic Acids

Biotechnology promises the ability to control biology and disease with laser-like precision, but biomolecules typically have poor cell penetration and unpredictable subcellular localization. Hundreds of peptides, proteins and nucleic acids are being developed as diagnostics and therapies. For many of these, intracellular delivery remains the primary obstacle. Currently, there are no quantitative, high-throughput tools to measure how much of a biomolecule enters a cell and where it distributes within the cell. Importantly, most widely-used methods for measuring cell penetration cannot rule out effects of material at the cell surface or trapped in endosomes.
We have devised a chloroalkane penetration assay (CAPA) that exclusively measures penetration to the cytoplasm. CAPA uses a cell line with stably expressed HaloTag in the cytoplasm, and measures the extent of covalent reaction between this enzyme and a small chloroalkane tag appended to the molecule of interest.

Joshua Kritzer
Associate Professor
Department of Chemistry
Tufts University

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Chemical, Computational, and Instrumental Tools for Challenging Peptide Syntheses

The Kay lab at the University of Utah is focused on D-peptide inhibitor development, which requires the chemical synthesis of mirror-image protein targets. Mirror-image proteins are not found in nature and are promising therapeutic agents due to their resistance to degradation by natural proteases. This webinar will describe the use of chemical, instrumental, and computational tools to overcome challenges associated with the chemical synthesis of large proteins via Fmoc solid-phase peptide synthesis and native chemical ligation. Specific examples will include the use of a reversible solubilizing tag (“helping hand”), pseudoproline dipeptides, UV monitoring, and an automated ligator program (“Aligator”) to predict the most efficient synthesis schemes.

Michael S Kay MD/PhD
Biochemistry, University of Utah

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