Work

Digital Microfluidics-Driven Cell-Free Protein Synthesis Platform Reveals Expression and Stability Determinants for Phytoglobins and Cysteine-to-Alanine Substituted Variants

Year: 2025

Type: article

Abstract: Heme proteins are central to metabolism and stress responses but remain challenging to express recombinantly due to cytotoxicity and folding constraints. Phytoglobins (Pgbs) exemplify these difficulties, as expression protocols often fail to translate across protein species. Here, we used a cell-free protein synthesis (CFPS) platform powered by digital microfluidics to screen expression determinants for sugar beet Pgb 1.2 (BvPgb 1.2), its C86A variant, and three of eight newly identified oat Pgbs (AsPgbs), including their cysteine-to-alanine substituted variants. Benchmarking with multiple solubility tags and cell-free blends revealed protein- and variant-specific preferences, with alanine substitutions frequently improving expression and purification yields. Oxidative additives such as glutathione disulfide, alone or combined with protein disulfide isomerase, consistently enhanced production, underscoring the importance of redox environments for Pgb stability. Two selected variants were scaled up and yielded putative soluble apo-form proteins. The results highlight how CFPS enables rapid, parallelized identification of expression requirements while uncovering the role of conserved cysteines and redox conditions in Pgb biogenesis.

Source: Antioxidants

DOI: 10.3390/antiox14111317

Authors: Leonard Groth, Leif Bülow

Institution: Lund University

Language: English

Topic: Hemoglobin structure and function

Subfield: Cell Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,499

Funders: European Molecular Biology Laboratory, Stiftelsen för Strategisk Forskning, Svenska Forskningsrådet Formas