High-throughput E. coli and pichia protein variant screening workflow

Overview

Defined Bioscience offers a modular, high-throughput workflow for expressing and screening recombinant protein variants. The platform uses E. coli for fast, economical screening and Komagataella phaffii (Pichia pastoris) as a complementary host for secreted or eukaryotic proteins. Variants are expressed in arrayed 96-well or deep-well plates, processed with magnetic bead-based affinity capture, and quantified for yield, purity, and solubility. For compatible targets, flow cytometry and cell sorting provide an even higher-throughput front end, enriching clones with desirable expression, display, ligand-binding, or affinity properties before arrayed production. This tiered approach moves inexpensive expression triage upstream, reducing hands-on time and per-candidate cost while enabling exploration of larger sequence spaces.

Technical specifications

Key features:

• Arrayed recombinant expression in 96-well and deep-well formats in both E. coli and K. phaffii
• Parallel magnetic bead-based affinity capture for rapid yield, purity, and solubility assessment
• Quantitative expression and QC readouts to rank variants before further functional testing
• Flow cytometry and cell sorting front end for targets compatible with fluorescent expression, display, ligand-binding, or affinity readouts
• Advancement of selected variants into biochemical or herbicide-relevant functional assays
• Modular workflow that can be tailored to variant number, host system, and screening criteria

The workflow is designed to identify variants with favorable expression, solubility, folding, and activity while minimizing effort spent producing poor-performing candidates.

Technology readiness level

The integrated workflow is at an early, proof-of-concept stage. The individual methods—microbial expression, magnetic bead purification, and flow cytometry—are well established, but the combined modular workflow has not yet been benchmarked against an existing screening campaign. Future validation includes selecting representative target proteins, optimizing expression and induction conditions, developing fluorescence-based enrichment readouts where compatible, and benchmarking throughput, success rate, hands-on time, and cycle time against current workflows. Pilot trials and co-development engagements are planned to refine the workflow and scale it to larger variant libraries.