New Peer-Reviewed Publication: Redox Control and Cysteine Substitutions Shape Phytoglobin Biogenesis
Cracking the Expression Bottleneck
Heme proteins (like phytoglobins) are vital but notoriously hard to express: heme cytotoxicity, folding constraints, and the need to balance redox conditions make protocols slow and brittle—what works for one protein often fails for another. The result is weeks of trial-and-error and low soluble yields. A peer-reviewed study from Lund University shows how cell-free protein synthesis (CFPS) on a digital-microfluidics platform rapidly maps expression requirements in parallel, short-circuiting those bottlenecks.
Innovation by Groth & Bülow
The team used Nuclera’s eProtein Discovery™ (digital-microfluidics CFPS) to screen phytoglobins and cysteine→alanine variants across solubility tags and redox/auxiliary additives—revealing protein- and variant-specific preferences. Oxidative additives (GSSG alone or PDI/GSSG) repeatedly boosted production, underscoring how redox control drives stability and yield. Notably, alanine substitutions often improved expression and purification, and several small tags (<10 kDa) outperformed larger ones.
What the team actually did
- Linear DNA → on-instrument expression: eGene™ constructs were designed in eProtein Discovery™ software and run on a digital microfluidics cartridge to screen 24 constructs against 8 cell-free blends (up to 192 unique conditions). Expression was quantified, with automated selection for on-chip purification.
- Condition space explored: solubility tags (e.g., P17, CUSF, FH8; untagged) × additives (e.g., GSSG, PDI/GSSG, TRXB1, DnaK mix, Zn²⁺, multi-cofactor mix, 3C protease). GSSG/PDI-GSSG dominated selected conditions, highlighting the role of oxidizing environments.
- Scale-up validation: two winners were scaled to 200 µL CFPS, purified, and characterized—~20–22 µM purified protein; mass photometry indicated monomers; nanoDSF showed Tm ≈ 55 °C for BvPgb 1.2.
The result?
Day-scale, parallel expression mapping, without chasing clones or hosts. In 24 hours, the platform delivered expression data for 192 condition combinations and on-chip purification for the best 30, pinpointing redox environments (GSSG/PDI-GSSG) as key levers, and showing how small solubility tags and cysteine→alanine substitutions can unlock yields. The study also produced soluble apo-forms of challenging phytoglobins and validated monomeric species on scale-up—evidence that digital-microfluidics CFPS can both accelerate protocol development and illuminate the biogenesis determinants behind hard proteins.
Who Gains the Most?
- Protein engineering & synbio teams: faster feasibility on intractable or redox-sensitive proteins
- Plant & ag-biotech labs: rapid multiplex screening across phytoglobins and related globins
- Core facilities/CROs: higher throughput screening with on-cartridge quantification and purification
- Method developers: a tractable path to test tags, blends, and additives systematically.
Dive Into the Study
Read the open-access paper: Digital Microfluidics-Driven Cell-Free Protein Synthesis Platform Reveals Expression and Stability Determinants for Phytoglobins and Cysteine-to-Alanine Substituted Variants (Antioxidants, 2025)
