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

Area of Science:

• Biotechnology
• Protein Engineering
• Molecular Biology

Background:

• Yeast display is a key tool in eukaryotic protein engineering.
• Conventional tags (e.g., FLAG, Myc) rely on antibody-antigen interactions, which can be unstable at low pH.
• Accurate quantification of yeast surface expression is crucial for protein engineering.

Purpose of the Study:

• To introduce and evaluate a SNAP tag for yeast display, overcoming low pH limitations.
• To compare SNAP tag performance against conventional antibody-based tags and direct fluorescent domain fusion.
• To demonstrate the utility of SNAP tagging for protein engineering in non-physiological pH environments.

Main Methods:

• Implemented a SNAP tag system on a yeast display platform.
• Utilized antibody fragment crystallizable (Fc) gene libraries for case studies.
• Compared SNAP tag performance with conventional peptide tags and direct fluorescent domain fusion at low pH.
• Quantified protein surface expression and enriched Fc variants with enhanced neonatal Fc receptor (FcRn) affinity.

Main Results:

• The SNAP tag enabled effective and pH-independent quantification of protein surface expression at low pH.
• Covalent SNAP tagging demonstrated superior performance compared to conventional antibody-based tags in low pH conditions.
• Enrichment of Fc variants with increased affinity to FcRn at pH 6.0 was successfully achieved using the SNAP tag.

Conclusions:

• Covalent SNAP tags overcome the pH-dependent limitations of conventional antibody-based yeast display tags.
• The SNAP tag facilitates precise protein display and quantification, enabling protein engineering applications outside of physiological pH.
• This method expands the scope of yeast display for engineering proteins under diverse environmental conditions.