1. Preparation of Site-Directed Mutagenesis Expression Vectors
Mutations are introduced into the PCR product by performing PCR on the original template using primers that contain the mutated sequence. After digesting the template plasmid with DpnI, the linearized template is religated into a circular form using a seamless ligation enzyme. The primary equipment involved includes a liquid handling workstation and a PCR machine.
2. High-throughput cell-free protein expression
Take a small aliquot of the circularized template containing the mutation, and perform PCR amplification of the linear template according to the recommended protocol. After adding the amplified linear template to the cell-free protein synthesis reaction mixture, carry out the cell-free reaction. Both liquid-handling steps can be automated using a high-throughput liquid handler; the subsequent incubation steps are performed on a PCR thermal cycler and either a constant‑temperature shaker or a constant‑temperature vortex mixer.
High-throughput screening model for site-directed mutagenesis of GFP
We performed site‑directed saturation mutagenesis at five positions within the active site of green fluorescent protein. We characterized the fluorescence properties of all mutant variants and identified the optimal structural configuration.
Within just over ten hours, we successfully constructed and expressed nearly a hundred site‑directed mutant proteins, followed by screening.
Using cell-free protein expression technology, the entire construction, expression, and screening workflow takes only 7 to 13 hours, with a total hands-on time of just 30 minutes. Compared with conventional methods, this approach not only significantly shortens the screening cycle and reduces labor costs but also substantially increases the number of proteins that can be screened.
