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Molecular & Cellular Proteomics 1:466-471, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

An Improved Protein Bioreactor

Efficient Product Isolation During in vitro Protein Biosynthesis Via Affinity Tag^*

Thorsten Lamla, Wolfgang Stiege and Volker A. Erdmann

From the Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.

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