Validation and Optimization of PURE Ribosome Display for Screening Synthetic Nanobody Libraries.

Abstract

Background/objectives: PURE (Protein synthesis Using Recombinant Elements), an ideal system for ribosome display, has been successfully used for nanobody selection. However, its limitations in nanobody selection, especially for synthetic nanobody libraries, have not been clearly elucidated, thereby restricting its utilization.

Methods: The PURE ribosome display selection process was closely monitored using RNA agarose gel electrophoresis to assess the presence of mRNA molecules in each fraction, including the flow-through, washing, and elution fractions. Additionally, a real-time validation method for monitoring each biopanning round was implemented, ensuring the successful enrichment of target protein-specific binders. The selection process was further optimized by introducing a target protein elution step prior to the EDTA-mediated disassembly, as well as by altering the immobilization surfaces. Finally, the efficiency of PURE ribosome display was enhanced by replacing the spacer gene.

Results: The efficiency of PURE ribosome display was merely 4% with an unfavourable spacer gene. Using this spacer gene, EGFP- and human fatty acid-binding protein 4-specific nanobodies from a synthetic nanobody library were we successfully identified through optimizing the selection process. Choosing a spacer gene less prone to secondary structure formation increased significantly its efficiency in displaying synthetic nanobody libraries.

Conclusions: Implementing a target protein elution step prior to EDTA-mediated disassembly and modifying the immobilization surfaces effectively increase selection efficiency. For PURE ribosome display, efficiency was further improved using a suitable spacer gene, enabling the display of large libraries.