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Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries
Cassandra Stowe1,2, Arnold Pizzey1, Tammy Kalber2
1Cancer Institute, Department of Haematology, Division of Medicine, University College London, London, United Kingdom.
Plos One
|November 5, 2015
Summary
Researchers developed a novel cell sorting platform for high-throughput protein screening. This technology enables rapid identification and characterization of proteins with desired properties from large libraries.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Protein engineering and design are crucial for developing novel functionalities.
- High-throughput screening (HTS) methods are essential for identifying valuable proteins from large libraries.
- Current HTS methods often require significant optimization and resources.
Purpose of the Study:
- To develop and evaluate a novel fluorescent activated cell sorting (FACS) based platform for high-throughput protein screening.
- To enable rapid, automated screening and analysis of protein libraries expressed in bacterial cells.
- To enhance the efficiency and throughput of identifying proteins with specific characteristics.
Main Methods:
- Development of a FACS platform for sorting single bacterial cells expressing protein libraries.
- Deposition of sorted cells onto solid media in a dense matrix format (44-195 colonies/cm²).
- Machine interrogation (<30s/plate) and bioinformatic analysis (~60s/plate) of deposited colonies.
- Spectral analysis using an optical imager to identify bioluminescent protein variants.
- Integration of a prototype electronic sort stream multiplexer to increase deposition rate.
Main Results:
- Demonstrated successful identification and characterization of a rare clone (0.5% population) expressing a bioluminescent protein.
- Achieved efficient machine interrogation and bioinformatic analysis of dense colony matrices.
- Increased colony deposition rate by 89.2% to 24 colonies per second with the multiplexer prototype.
- Validated the platform's capability for spectrally analyzing and isolating specific bacterial clones.
Conclusions:
- The developed FACS-based screening platform enables high-throughput identification of proteins with novel or refined characteristics.
- The dense matrix format and automated analysis facilitate rapid screening of large protein libraries.
- This technology represents a significant advancement with the potential to form the basis of next-generation HTS for protein engineering.
