Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems

PLoS One. 2022 Mar 17;17(3):e0265274. doi: 10.1371/journal.pone.0265274. eCollection 2022.

Abstract

Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biosensing Techniques*
  • Cell-Free System
  • Protein Biosynthesis
  • Proteins
  • Quantum Dots*

Substances

  • Proteins

Grants and funding

The authors acknowledge the Office of Naval Research (ONR), the U.S. Naval Research Laboratory (NRL), and the NRL Nanoscience Institute for funding support. S.L.H. acknowledges an NRC Fellowship through NRL. I.L.M. and G.A.E acknowledge the National Institute of Food and Agriculture, U.S. Department of Agriculture, under Award #2020-67021-31254, and the Strategic Environmental Research and Development Program (SERDP), under Award # WP21-1073 New Start Project (W74RDV03497375). The funders had no role in study design, data collection and analysis, or preparation of the manuscript.