The covalent SNAP tag for protein display quantification and low-pH protein engineering.

Yeast display has become an important tool for modern biotechnology with many advantages for eukaryotic protein engineering. Antibody-based peptide interactions are often used to quantify yeast surface expression (e.g., by fusing a target protein to a FLAG, Myc, polyhistidine, or other peptide tag). However, antibody-antigen interactions require high stability for accurate quantification, and conventional tag systems based on such interactions may not be compatible with a low pH environment. In this study, a SNAP tag was introduced to a yeast display platform to circumvent disadvantages of conventional antibody display tags at low pH. SNAP forms a covalent bond with its small-molecule substrate, enabling precise and pH-independent protein display tagging. We compared the SNAP tag to conventional antibody-based peptide fusion and to direct fluorescent domain fusion using antibody fragment crystallizable (Fc) gene libraries as a case study in low pH protein engineering. Our results demonstrated that covalent SNAP tags can effectively quantify protein-surface expression at low pH, enabling the enrichment of Fc variants with increased affinity at pH 6.0 to the neonatal Fc receptor (FcRn). Incorporation of a covalent SNAP tag thus overcomes disadvantages of conventional antibody-based expression tags and enables protein-engineering applications outside of physiological pH.

Ultrasonically-guided flow focusing generates precise emulsion droplets for high-throughput single cell analyses.

Emulsion-based techniques have dramatically advanced our understanding of single-cell biology and complex single-cell features over the past two decades. Most approaches for precise single cell isolation rely on microfluidics, which has proven highly effective but requires substantial investment in equipment and expertise that can be difficult to access for researchers that specialize in other areas of bioengineering and molecular biotechnology. Inspired by the robust droplet generation technologies in modern flow cytometry instrumentation, here we established a new platform for high-throughput isolation of single cells within droplets of tunable sizes by combining flow focusing with ultrasonic vibration for rapid and effective droplet formation. Application of ultrasonic pressure waves to the flowing jet provided enhanced control of emulsion droplet size, permitting capture of 25,000 to 50,000 single cells per minute. As an example application, we applied this new droplet generation platform to sequence the antibody variable region heavy and light chain pairings (VH:VL) from large repertoires of single B cells. We demonstrated the recovery of > 40,000 paired CDRH3:CDRL3 antibody clusters from a single individual, validating that these droplet systems can enable the genetic analysis of very large single-cell populations. These accessible new technologies will allow rapid, large-scale, and precise single-cell analyses for a broad range of bioengineering and molecular biotechnology applications.