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Engineering Ag43 Signal Peptides with Bacterial Display and Selection
Darius Wen-Shuo Koh1, Jian-Hua Tay1, Samuel Ken-En Gan1,2,3,4
1Antibody & Product Development Laboratory, Agency for Science, Technology, and Research (A*STAR), Singapore 138671, Singapore.
Methods and Protocols
|January 17, 2023
Summary
Researchers enhanced protein surface display in Escherichia coli using error-prone PCR and single-cell sorting. This method rapidly improves microbial systems for applications in medicine and biocatalysis.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Protein display and secretion in prokaryotes are crucial for engineered microbial applications.
- Optimizing signal peptides enhances protein surface display efficiency.
Purpose of the Study:
- To develop a high-throughput method for selecting improved signal peptides for Escherichia coli protein display.
- To augment bacterial surface display using error-prone polymerase chain reaction (epPCR) and single-cell sorting.
Main Methods:
- Utilized epPCR to generate random mutations in Ag43 signal peptides.
- Employed single-cell sorting and microplate assays for selection.
- Verified surface protein display via trypsinization and SDS-PAGE.
Main Results:
- Isolated clones with 1.4- to 3-fold increased surface display of sfGFP-beta-lactamase.
- Demonstrated successful extracellular protein display and release.
- Achieved significant improvements in display after three rounds of mutagenesis and selection.
Conclusions:
- The developed workflow offers a rapid and high-throughput approach for enhancing bacterial surface display.
- This method can be applied to optimize other bacterial proteins for various biotechnological applications.
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