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A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...
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High-throughput Protein Expression Generator Using a Microfluidic Platform
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mRNA Display Pipeline for Protein Biosensor Construction.

Zhenling Cui1,2,3, Cagla Ergun Ayva2,3, Yi Jin Liew4

  • 1ARC Centre of Excellence in Synthetic Biology, Brisbane, Queensland 4001, Australia.

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|May 29, 2024
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Summary

Developing modular protein biosensors is streamlined by a new workflow. This method rapidly generates specific binders, improving biosensor assembly and prototyping for liver toxicity markers.

Keywords:
DARPinsMonobodiesmRNA displayprotein biosensorsynthetic protein binding domain

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Engineering

Background:

  • Protein biosensor development is often inefficient, relying on trial-and-error methods.
  • Modular biosensor designs offer a promising avenue for creating adaptable biosensors.
  • The integration of specific binding domains into biosensor architectures is crucial for target recognition.

Purpose of the Study:

  • To establish and validate a high-throughput workflow for generating specific binders for protein biosensors.
  • To develop novel biosensors for detecting liver toxicity markers using the established workflow.
  • To investigate the relationship between binder affinity and biosensor performance.

Main Methods:

  • Utilized mRNA display and ribosome display for selection of hyper-stable monobody domains and DARPins, respectively.
  • Integrated selected binders into a two-component allosteric biosensor architecture featuring a calmodulin-reporter chimera.
  • Tested the workflow by developing biosensors for liver toxicity markers: aspartate aminotransferase (cytosolic and mitochondrial) and alanine aminotransferase 1.

Main Results:

  • The workflow consistently generated over 10^3 unique binders per target within one week.
  • Binder affinity for the target did not directly correlate with biosensor performance.
  • Interactions between binding domains and the reporter module significantly influenced biosensor activity and dynamic range.

Conclusions:

  • The developed workflow offers an efficient method for generating specific binders for protein biosensor construction.
  • Multiplexed biosensor assembly and prototyping are critical steps for optimizing biosensor function post-binding domain selection.
  • This approach holds promise for accelerating the development of custom protein biosensors for various applications, including diagnostics.