Effects of growth rate on cell extract performance in cell-free protein synthesis
Article first published online: 3 MAY 2006
Copyright © 2006 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 94, Issue 4, pages 618–624, 5 July 2006
How to Cite
Zawada, J. and Swartz, J. (2006), Effects of growth rate on cell extract performance in cell-free protein synthesis. Biotechnol. Bioeng., 94: 618–624. doi: 10.1002/bit.20831
- Issue published online: 29 MAY 2006
- Article first published online: 3 MAY 2006
- Manuscript Accepted: 6 DEC 2005
- Manuscript Received: 14 AUG 2005
- cell-free protein synthesis;
- growth rate;
- high cell density fermentation
Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity. © 2006 Wiley Periodicals, Inc.