Premium
Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell‐Free Protein Synthesis
Author(s) -
Jewett Michael C.,
Swartz James R.
Publication year - 2008
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0341693
Subject(s) - cell free protein synthesis , chromatography , elution , chemistry , yield (engineering) , cell free system , protein biosynthesis , chloramphenicol , biochemistry , specific activity , affinity chromatography , active site , enzyme , materials science , metallurgy , antibiotics
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.