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.
Jewett
Michael C
MC
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Swartz
James R
JR
D000255:Adenosine Triphosphate / Q000737:chemistry; D002474:Cell-Free System / Q000737:chemistry*; D015500:Chloramphenicol O-Acetyltransferase / Q000138:chemical synthesis* / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods*; D004789:Enzyme Activation; D005240:Feasibility Studies; D008832:Microchemistry / Q000379:methods*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011771:Pyruvate Oxidase / Q000737:chemistry*
D000255:Adenosine Triphosphate; D011771:Pyruvate Oxidase; D015500:Chloramphenicol O-Acetyltransferase