Spatiotemporal Control Over Protein Release from Artificial Cells via a Light-Activatable Protease

Arjan Hazegh Nikroo, Wiggert J. Altenburg, Thijs W. van Veldhuisen, Luc Brunsveld, Jan C.M. van Hest (Corresponding author)

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

The regulation of protein uptake and secretion by cells is paramount for intercellular signaling and complex multicellular behavior. Mimicking protein-mediated communication in artificial cells holds great promise to elucidate the underlying working principles, but remains challenging without the stimulus-responsive regulatory machinery of living cells. Therefore, systems to precisely control when and where protein release occurs should be incorporated in artificial cells. Here, a light-activatable TEV protease (LaTEV) is presented that enables spatiotemporal control over protein release from a coacervate-based artificial cell platform. Due to the presence of Ni2+-nitrilotriacetic acid moieties within the coacervates, His-tagged proteins are effectively sequestered into the coacervates. LaTEV is first photocaged, effectively blocking its activity. Upon activation by irradiation with 365 nm light, LaTEV cleaves the His-tags from sequestered cargo proteins, resulting in their release. The successful blocking and activation of LaTEV provides control over protein release rate and triggerable protein release from specific coacervates via selective irradiation. Furthermore, light-activated directional transfer of proteins between two artificial cell populations is demonstrated. Overall, this system opens up avenues to engineer light-responsive protein-mediated communication in artificial cell context, which can advance the probing of intercellular signaling and the development of protein delivery platforms.

Original languageEnglish
Article numbere2400353
JournalAdvanced Biology
VolumeXX
Issue numberX
Early online date27 Sept 2024
DOIs
Publication statusE-pub ahead of print - 27 Sept 2024

Keywords

  • Green Fluorescent Proteins
  • Protein Engineering
  • Coacervates
  • Protease
  • artificial cells
  • Liquid-liquid phase separation
  • scaffold proteins
  • TEV protease
  • photoswitchable proteins
  • photoactivation
  • UV irradiation
  • synthetic signaling
  • protein release
  • coacervates
  • liquid-liquid phase separation

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