Cell Free Protein Expression Optimization

URL: https://cloud.ginkgo.bio/protocols/cell-free-protein-expression-optimization Status: Ginkgo Certified Price: $199/sample Turnaround: up to 11 days

Overview

Design of Experiment (DoE) approach to expressing protein targets in a proprietary reconstituted E. coli transcription-translation system. Each construct is evaluated in up to 24 reaction conditions per protein, including target-specific additives such as chaperones, disulfide-bond enhancers, and cofactors. Designed for difficult-to-express proteins including membrane proteins and targets with disulfide or cofactor requirements.

Input

• DNA sequence in .fasta format

Output

• Comparative Yield: Titer data mapped across all tested variables (lysates, temps, additives)
• Purity Profiling: Target protein vs. background impurities to find highest quality yield
• Optimal Conditions: Overlaid electropherograms pinpointing the exact formulation for a given sequence

Automated Workflow

Phase 1 - Reagent Prep

1. Retrieve plates from 4 deg C
2. Thaw at room temperature
3. PBS backfill

Phase 2 - CFPS Reaction Setup & Incubation

1. Retrieve plates from 4 deg C
2. Dispense lysate
3. QC plate read
4. Incubate (shaking or static, condition-dependent)

Phase 3 - Quantification Prep & Read

1. Dispense PBS
2. Unseal plate
3. LabChip quantification
4. Seal plate
5. Store at 4 deg C

Protocol Parameters

• Payloads & Reagents
• Bravo Stamp
• HiG Centrifuge
• Incubation & Storage

Optimization Variables

The DoE matrix can span up to 24 conditions per protein, varying:

• Lysate composition (different E. coli extract formulations)
• Temperature (incubation temperature profiles)
• Additives:
• Chaperones (for folding-challenged targets)
• Disulfide-bond enhancers (for targets requiring disulfide bridges)
• Cofactors (metal ions, coenzymes, prosthetic groups)
• Other target-specific supplements

Ordering

• Number of Proteins: configurable
• Number of Replicates: configurable
• File Upload: CSV, Excel, FASTA, TXT, PDF, ZIP
• Additional Details: free-text field for special requirements

Certification Milestones

• Dry Run Complete
• Wet Run Complete
• Biovalidation Complete
• App Note Complete

Use Cases

• Optimizing expression of difficult-to-express proteins
• Membrane protein expression screening
• Identifying optimal conditions for disulfide-bonded proteins
• Cofactor-dependent protein expression
• Systematic exploration of expression parameter space
• Finding the best formulation before scaling up production