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High-Throughput Screening Case: Cell-Free Expression Screening Based on a Mutant Library—phi29 DNA Polymerase
Release date:
2023-12-08
Abstract
Enzymes are crucial functional proteins in nature, and their catalytic activity plays an important role in areas such as gene editing and stem cell technologies, the production of targeted therapeutics, the food industry, the textile industry, medical diagnostics, and drug synthesis. In this study, using phi29 DNA polymerase as a representative, we developed a high-throughput enzyme‑screening platform based on cell‑free protein expression. Our results demonstrate that, within three days, we successfully expressed 96 phi29 DNA polymerase mutants and assessed their activities, identifying several highly efficient mutant variants.
In recent years, driven by advances in AI technology, AI‑assisted protein design has been steadily becoming a reality. Within the AI‑driven cycle of design–construction–testing–learning, both construction and testing play a crucial role in fostering AI’s development.
The typical conventional protein expression workflow is as follows:
After constructing the mutant gene using a semi‑rational approach, the construct is cloned into a plasmid, which is then transformed into the host cells. Single colonies are picked from the plated culture and propagated to obtain cell lines that express the mutant protein. By scaling up these cell lines and subsequently lysing the cells, a crude mixture containing the target mutant protein can be isolated. This mixture can then be subjected to purification and functional assays. However, this method is technically demanding and time‑consuming; moreover, if the target mutant protein is toxic to the host cells, expression may fail.
For such high-throughput screening, cell-free protein expression is clearly a more efficient choice. This technology leverages the cellular machinery of lysed organisms—such as ribosomes and transcription‑translation enzymes—and incorporates an appropriate cofactor system, including energy sources and amino acids. By adding a template DNA, it enables rapid and efficient protein synthesis in vitro. Cell-free protein expression significantly streamlines the protein‑production workflow, making it better suited to high‑throughput applications. Moreover, its rapid expression kinetics reduce the time required for protein production, thereby shortening overall project timelines.
In this study, using phi29 DNA polymerase as a model, we employed cell-free protein expression to efficiently produce 96 protease mutants within three days and assess their enzymatic activities. Our findings highlight the potential of cell-free systems for high-throughput screening of proteases and drug candidates.
Method
Mutant Introduction and Cyclization
We selected the 221st and 350th amino acids of phi29 DNA polymerase as mutation sites. First, we introduced the mutations into the target fragment using PCR with specific primers; then, we incorporated these mutated fragments via overlap PCR. Finally, we ligated the resulting gene fragment together with a linearized circular fragment using a seamless ligation enzyme, thereby generating a circular plasmid.
During this process, after each PCR reaction using the plasmid as a template, DpnI enzyme was used to digest the sample at 37°C for 1 hour to degrade the original template.
Plasmid transformation and plating
Add 5 μL of the mutant plasmid to 100 μL of DH5α competent cells, gently mix, and incubate on ice for 30 minutes. Then, place the competent cells in a 42°C water bath, heat-shock for 90 seconds, and promptly transfer them back to ice; allow to stand for 3–5 minutes. Next, add the cells to 500 μL of antibiotic-free SOC or LB medium, mix gently, and shake at 37°C for 1 hour. Subsequently, centrifuge the culture (5,000 rpm, 1 minute) to pellet the cells, discard most of the supernatant, resuspend the pellet in approximately 50–100 μL of medium, and finally spread the suspension evenly onto LB plates containing kanamycin. Incubate overnight at 37°C.
Pick single colonies and preserve the strains.
Add 100 μL of LB medium containing kanamycin to a 96-well plate, then pick single colonies from the LB agar plate and transfer them into the 96-well plate. Incubate at 37°C on a shaker at 180 rpm for 1 hour.
Bacterial PCR and High-Throughput Protein Expression
From each well of a 96-well plate subjected to shaking culture for 1 hour, 1 μL of culture medium was withdrawn and used as a template in the PCR reaction. Following an appropriate PCR protocol, 5 μL of the PCR reaction product was then added as a template to a cell-free expression system. After 4 hours of incubation, the target protein enzyme—phi29 DNA polymerase—was produced within the system.
Activity assay
The activity assay of phi29 DNA polymerase involves using the enzyme’s polymerase activity to amplify a linear template of the green fluorescent protein gene, with the fluorescence intensity of the final product serving as an indicator of phi29 DNA polymerase activity. First, 1 μL of the supernatant from the cell-free reaction mixture is directly collected as the enzyme solution and added to an amplification system containing a buffer that activates phi29, a plasmid template encoding the green fluorescent protein gene, and the primers required for amplification. The mixture is then incubated at 42°C for 2 hours to generate the linear template. Finally, 5 μL of this linear template is transferred into a cell-free reaction system with a final volume of 50 μL; after 6 hours, the fluorescence signal is measured to identify phi29 DNA polymerase mutants exhibiting enhanced activity.
Result
Active result
Under excitation at 485 nm and emission at 535 nm, the fluorescence intensity of green fluorescent protein was measured. The fluorescence values varied significantly among different wells, indicating that the mutant forms of phi29 DNA polymerase exhibit differing levels of enzymatic activity.
Mutation sequencing
After determining the enzymatic activity, we selected the bacterial cultures corresponding to the highest‑activity wells from the previously stored 96‑well plate, expanded them, and proceeded with sequencing. We successfully identified the top three phi29 DNA polymerase mutants in terms of activity on this plate. The amino acid substitutions at positions 221 and 350 are as follows.
Conclusion
Through the aforementioned experiments, Perotin Bio has demonstrated a method for high-throughput screening and validation of mutant proteins using cell-free protein expression technology. This approach enabled the construction, expression, and activity assessment of nearly one hundred mutant proteins within three days, significantly reducing protein‑screening timelines, lowering manual‑operation costs, and enhancing R&D efficiency.
The continuous enhancement of protein bioactivity and stability plays a pivotal role in the widespread adoption of protein‑based products across sectors such as healthcare, agriculture, papermaking, textiles, bioenergy, and personal care. By leveraging cell‑free protein expression technologies, protein engineering and screening can be carried out more efficiently and rapidly, shortening the R&D timelines for enzyme preparations, therapeutic proteins, and other protein‑based products while reducing development costs, thereby accelerating the advancement of novel recombinant proteins, enzymes, antibodies, and other protein‑based innovations.
As a specialized biotechnology company focused on cell-free protein expression, Perotin Bio will continue to leverage the advantages of this technology to develop an even broader range of applications.
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