Digital Microfluidics-Driven Cell-Free Protein Synthesis Platform Reveals Expression and Stability Determinants for...

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Heme proteins, crucial for metabolism and stress, are difficult to express recombinantly due to toxicity and folding issues.
• Phytoglobins (Pgbs) present unique expression challenges, with protocols often failing to transfer between species.
• Optimizing Pgb expression requires understanding specific protein and variant requirements.

Purpose of the Study:

• To employ a cell-free protein synthesis (CFPS) platform for rapid screening of expression determinants for sugar beet and oat phytoglobins.
• To identify optimal conditions, including solubility tags, cell-free blends, and oxidative additives, for enhanced Pgb production.
• To investigate the role of conserved cysteines and redox environments in Pgb biogenesis.

Main Methods:

• Utilized a digital microfluidics-powered CFPS platform for high-throughput screening of expression variables.
• Benchmarked various solubility tags and CFPS reaction mixtures for different Pgb variants.
• Assessed the impact of oxidative additives (glutathione disulfide, protein disulfide isomerase) on Pgb production.

Main Results:

• Identified protein- and variant-specific preferences for expression and purification conditions.
• Demonstrated that alanine substitutions at cysteine residues frequently improved expression and purification yields.
• Showcased consistent enhancement of Pgb production with oxidative additives, highlighting the importance of redox environments.
• Successfully scaled up two selected variants, yielding soluble apo-form proteins.

Conclusions:

• CFPS is an effective tool for rapid, parallelized identification of optimal protein expression requirements.
• Conserved cysteines and specific redox conditions play a critical role in phytoglobin biogenesis and stability.
• The study provides a framework for optimizing the recombinant expression of challenging heme proteins like phytoglobins.