Cell-Free Protein Synthesis With Prokaryotic Combined Transcription-Translation
Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems. We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.
Key WordsProkaryotic combined transcription-translation S30 extract cell-free protein synthesis in vitro chloramphenicol acetyl transferase T7 bacteriophage RNA polymerase protein purification
- 1.Jewett, M. C., Voloshin, A., and Swartz, J. R. (2002) Prokaryotic systems for in vitro expression, in Gene Cloning and Expression Technologies (Weiner, M. P., and Lu, Q., eds.), Eaton Publishing, Westborough, MA, pp. 391–411.Google Scholar
- 12.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
- 14.Pratt, J. M. (1984) Coupled transcription-translation in prokaryotic cell-free systems, in Transcription and Translation: A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL Press, New York, NY, pp. 179–209.Google Scholar
- 17.Jewett, M. C. and Swartz, J. R. (2004) Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis. Biotechnol. Prog. In press.Google Scholar
- 19.Michel-Reydellet, N., Calhoun, K. A., and Swartz, J. R. (2004) Amino acid stabilization for cell-free protein synthesis by modification of the E. coli genome. Metabolic Engineering. In press.Google Scholar