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High-throughput Protein Expression Generator Using a Microfluidic Platform
Published on: August 23, 2012
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Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering
Matthew C Blackburn1, Ekaterina Petrova1, Bruno E Correia1
1Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Nucleic Acids Research
|December 26, 2015
Summary
We developed a rapid gene synthesis and protein expression method (APE-MITOMI) to accelerate protein design. This approach allows for the characterization of hundreds of proteins in just days, advancing synthetic biology and medicine.
Area of Science:
- Biotechnology
- Synthetic Biology
- Protein Engineering
Background:
- Synthetic genes are increasingly available, but integrated methods for rapid protein design and characterization are lacking.
- Accelerating the design-to-characterization pipeline is crucial for advancing medicine, biotechnology, and synthetic biology.
Purpose of the Study:
- To develop an integrated, rapid approach for protein design, synthesis, expression, and characterization.
- To reduce the time from protein design to quantitative characterization significantly.
Main Methods:
- Developed a solid-phase gene synthesis method based on asymmetric primer extension (APE).
- Coupled APE directly to high-throughput, on-chip protein expression, purification, and characterization using mechanically induced trapping of molecular interactions (MITOMI).
- Circumvented traditional molecular cloning and cell-based steps.
Main Results:
- The APE-MITOMI method reduces the protein design-to-characterization time to 3-4 days.
- Successfully synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TFs).
- Demonstrated that while ZF TFs can be engineered for DNA sequence recognition, precise binding energy landscape engineering remains challenging; however, ZF-DNA affinity can be engineered independently of sequence specificity.
Conclusions:
- APE-MITOMI is a generic and rapid approach facilitating protein biophysics studies and protein design/engineering.
- In silico modeling can explain observed differences in protein-DNA affinity.
- The study highlights challenges and possibilities in engineering precise protein-DNA interactions for synthetic biology applications.
