Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell‐Free Protein Synthesis

Two strategies for ATP regeneration during cell‐free protein synthesis were applied to the large‐scale production and single‐column purification of active chloramphenicol acetyl transferase (CAT). Fed‐batch reactions were performed on a 5–10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 ± 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed‐batch cell‐free protein synthesis reaction.