In Vitro and In Vivo Analysis of Extracellular Vesicle-Mediated Metastasis Using a Bright, Red-Shifted Bioluminescent Reporter Protein.

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. This study focuses on a novel method to image EV subtypes and their biodistribution in vivo. A red-shifted bioluminescence resonance energy transfer (BRET) EV reporter is developed, called PalmReNL, which allows for highly sensitive EV tracking in vitro and in vivo.

Ancestral Sequence Reconstruction and Alternate Amino Acid States Guide Protein Library Design for Directed Evolution.

Engineered proteins possess nearly limitless possibilities in medical and industrial applications but finding a precise amino acid sequence for these applications is challenging. A robust approach for discovering protein sequences with a desired functionality uses a library design method in which combinations of mutations are applied to a robust starting point. Determining useful mutations can be tortuous, yet rewarding; in this chapter, we present a novel library design method that uses information provided by ancestral sequence reconstruction (ASR) to create a library likely to have stable proteins with diverse function.

Site-wise Diversification of Combinatorial Libraries Using Insights from Structure-guided Stability Calculations.

Many auspicious clinical and industrial accomplishments have improved the human condition by means of protein engineering. Despite these achievements, our incomplete understanding of the sequence-structure-function relationship prevents rapid innovation. To tackle this problem, we must develop and integrate new and existing technologies.