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An “Accelerator” for Directed Enzyme Evolution: A High-Throughput Screening Strategy Based on CFPS (Part I)
Release date:
2024-10-11
Enzymes are crucial functional proteins in nature, and their catalytic activity plays a pivotal role in fields such as gene editing and stem cell technologies, the production of targeted therapeutics, the food industry, the textile industry, medical diagnostics, and drug synthesis. However, due to limitations in thermal stability, pH tolerance, solvent compatibility, substrate specificity, and catalytic efficiency, natural enzymes are often not the optimal choice for industrial applications. Directed evolution is a widely used approach to enhance enzyme catalytic performance and thermal stability.
The mutant protein screening workflow based on cell‑expression systems comprises steps such as constructing a mutagenized gene library, generating expression vectors, transformation, single‑colony picking, scale‑up cultivation, cell lysis, purification, and activity assays. These procedures are labor‑intensive, time‑consuming, and costly in terms of manpower. Moreover, if the target mutant protein is toxic to the host cells, it may even lead to expression failure. Therefore, more efficient high‑throughput screening strategies are urgently needed for mutant protein selection.
Cell-free protein synthesis (CFPS) is an in vitro protein biosynthesis technology that does not involve living cells, offering distinct advantages in high-throughput screening applications:
① CFPS is easy to operate and can be integrated with high-throughput liquid-handling workstations to enable automated workflows.
②CFPS is an open reaction system that allows adjustment of reaction conditions such as pH and temperature to suit specific proteins, and enables direct assessment of mutant protein function in the post-reaction supernatant without the need for purification.
③ CFPS is not constrained by host cell viability, enabling the production of cytotoxic proteins.
④ CFPS permits the use of PCR products as templates, significantly shortening the template preparation workflow.
PLD High-Throughput Screening Case Studies
This paper presents a high-throughput enzyme screening platform developed by Perotin Bio, built upon its proprietary CFPS system:
Mutagenesis libraries were constructed by introducing mutations via degenerate primers. Individual mutant genes were isolated from colonies, and linear templates were obtained through colony PCR. These linear templates were directly used in CFPS reactions, yielding mutant enzymes within a few hours; enzyme activity was then assessed by analyzing the supernatant.
Using this approach, we successfully expressed 96 phi29 DNA polymerase mutants within three days and assessed their enzymatic activities, identifying several highly efficient phi29 DNA polymerase variants.
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 integrated the mutated fragments via overlap PCR. Finally, we ligated the assembled gene fragment with the linearized circular fragment using a seamless ligation enzyme to generate 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 transfer the competent cells to a 42°C water bath, heat-shock for 90 seconds, and promptly return them 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. 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 shake‑incubation 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 product was then added as a template to a cell‑free expression system. After 4 hours of incubation, the target protein—phi29 DNA polymerase—was produced in the reaction mixture.
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 enzyme’s activity assessed by the intensity of the resulting green fluorescence. First, 1 μL of the supernatant from the final 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 of 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 intensity is measured to identify phi29 DNA polymerase mutants with enhanced activity.
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 catalytic 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.
Summary
Through the experiments described above, we have demonstrated a high-throughput screening and validation approach for mutant enzymes based on the Perrotin cell-free protein expression system. This protocol enabled the construction, expression, and activity assessment of nearly one hundred mutant proteins within three days, substantially reducing the time required for protein screening and lowering labor‑intensive costs, thereby significantly enhancing R&D efficiency.
In addition to the approach presented in this paper, Perotin Bio has also developed a high-throughput screening method that introduces mutations via primers and directly generates linear templates by PCR, enabling the screening of nearly one hundred GFP mutants within a single day. The detailed protocol will be described in a forthcoming article—stay tuned!
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