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Mimicking the Escherichia coli cytoplasmic environment activates long‐lived and efficient cell‐free protein synthesis
Author(s) -
Jewett Michael C.,
Swartz James R.
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20026
Subject(s) - cell free protein synthesis , escherichia coli , cell free system , protein biosynthesis , biochemistry , cytoplasm , adenosine triphosphate , phosphate , chemistry , enzyme , reagent , metabolic engineering , gene
Abstract Cell‐free translation systems generally utilize high‐energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell‐free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The “Cytomim” system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 μg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli , the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high‐energy phosphate compounds. © 2004 Wiley Periodicals, Inc.