Polycistronic cell-free system for high-throughput variant screening

Overview

Biocatalist is developing a polycistronic cell-free protein expression and screening platform that multiplies the throughput of variant screening. Instead of expressing and testing one protein variant per reaction, the system encodes several variants in a single polycistronic gene, separated by cleavable linkers. After cell-free expression, the resulting polyprotein is purified, cleaved, and separated into individual variants for activity screening. This approach is intended to accelerate enzyme and protein engineering workflows by 5- to 8-fold, allowing roughly 100 cell-free reactions to screen 500–800 variants in about four days.

Technical specifications

How it works:

• Polycistronic design: A single gene construct encodes 5–8 distinct protein variants separated by unique linker sequences, enabling each cell-free reaction to produce multiple candidates simultaneously.
• Cell-free expression: In vitro transcription-translation (IVTT) produces micro- to milligram quantities of protein in hours, avoiding cell cloning and culture bottlenecks. The system supports proteins larger than 250 kDa and can accommodate post-translational modifications.
• Purification and cleavage: Expressed polyproteins are His-tag purified and then treated with a protease (TEV protease or caspase) that cuts the linkers and releases individual variants into one mixture.
• Automated fractionation: The variant mixture is resolved by automated ion-exchange HPLC and collected into 96-well plates, using the distinct biochemical properties of each linker to obtain separate elution profiles.
• Activity screening: Fractions are screened for enzymatic activity, mapping elution peaks back to candidate performance.
• Optional direct pooling: If purification is not required, unlinked variant genes can be pooled directly in each reaction before HPLC fractionation, simplifying the workflow.

Applications:

• Enzyme engineering and directed evolution
• High-throughput screening of protein variants
• Biocatalyst development for pharmaceutical, agricultural, and industrial applications

Technology readiness level

The platform is currently at an early development stage. The proposed validation plan is organized in three phases: (1) design and expression of polycistronic constructs containing 5–8 variants; (2) protease cleavage and automated HPLC fractionation; and (3) enzymatic activity screening of isolated fractions. Completion of these phases is intended to demonstrate the 5- to 8-fold throughput multiplier in practice. As a result, the technology is not yet commercially deployed and should be considered a pre-commercial R&D offering.