4.7 Article

Generalized Approach towards Secretion-Based Protein Production via Neutralization of Secretion-Preventing Cationic Substrate Residues

Journal

Publisher

MDPI
DOI: 10.3390/ijms23126700

Keywords

ABC transporter; type I secretion system (T1SS); protein secretion; recombinant protein expression; protein mutagenesis

Funding

  1. Global Frontier Project from the Intelligent Synthetic Biology Center
  2. Korea Science Academy of KAIST
  3. Ministry of Science and ICT of the Republic of Korea

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This study provides a method to rescue secretion by correcting secretion-blocking regions, making a significant step in understanding the physiological properties of ABC transporter-dependent protein secretion and laying the foundation for the development of a secretion-based protein-producing platform.
Many heterologous proteins can be secreted by bacterial ATP-binding cassette (ABC) transporters, provided that they are fused with the C-terminal signal sequence, but some proteins are not secretable even though they carry the right signal sequence. The invention of a method to secrete these non-secretable proteins would be valuable both for understanding the secretory physiology of ABC transporters and for industrial applications. Herein, we postulate that cationic supercharged regions within the target substrate protein block the secretion by ABC transporters. We also suggest that the secretion of such substrate proteins can be rescued by neutralizing those cationic supercharged regions via structure-preserving point mutageneses. Surface-protruding, non-structural cationic amino acids within the cationic supercharged regions were replaced by anionic or neutral hydrophilic amino acids, reducing the cationic charge density. The examples of rescued secretions we provide include the spike protein of SARS-CoV-2, glutathione-S-transferase, streptavidin, lipase, tyrosinase, cutinase, growth factors, etc. In summary, our study provides a method to predict the secretability and a tool to rescue the secretion by correcting the secretion-blocking regions, making a significant step in understanding the physiological properties of ABC transporter-dependent protein secretion and laying the foundation for the development of a secretion-based protein-producing platform.

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