Rapid screening of staphylokinase protein variants using an unpurified cell‐free expression system

Protein engineering approaches, including rational design and directed evolution, are essential for optimizing protein properties in biotechnology. However, their application is often limited by the need to experimentally characterize large numbers of variants. This is especially true for directed evolution, where selected candidates require heterologous expression and purification before functional testing. To reduce this bottleneck for staphylokinase, a fibrin-specific thrombolytic with therapeutic potential, we developed a rapid screening platform based on cell-free protein synthesis (CFPS) and a chromogenic plasminogen-activation assay. Activity rankings from crude CFPS mixtures closely matched those from purified proteins, showing that the method provides reliable functional readouts without purification. This CFPS-based workflow offers a fast, scalable, and efficient solution for early-stage screening of staphylokinase variants and can accelerate the identification of new thrombolytic candidates. The modularity of this method also allows its facile adjustment for other enzymes.