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

Michael C. Jewett, James R. Swartz*

*Corresponding author for this work

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)102-109
Number of pages8
JournalBiotechnology Progress
Volume20
Issue number1
DOIs
StatePublished - Jan 2004

ASJC Scopus subject areas

  • Biotechnology

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