Document Detail

Streamlining Escherichia coli S30 extract preparation for economical cell-free protein synthesis.
MedLine Citation:
PMID:  15801786     Owner:  NLM     Status:  MEDLINE    
Escherichia coli extracts activate cell-free protein synthesis systems by providing the catalysts for translation and other supporting reactions. Recent results suggest that high-density fermentations can be used to provide the source cells, but the subsequent cell extract preparation procedure requires multiple centrifugation and dialysis steps as well as an expensive runoff reaction. In the work reported here, the extract preparation protocol duration was reduced by nearly 50% by significantly shortening several steps. In addition, by optimizing the runoff incubation, overall reagent costs were reduced by 70%. Nonetheless, extracts produced from the shorter, less expensive procedure were equally active. Crucial steps were further examined to indicate minimal ribosome loss during the standard 30,000g centrifugations. Furthermore, sucrose density centrifugation analysis indicated that although an incubation step significantly activates the extract, ribosome/polysome dissociation is not required. These insights suggest that consistent cell extract can be produced more quickly and with considerably less expense for large-scale cell-free protein production, especially when combined with high-density fermentation protocols.
David V Liu; James F Zawada; James R Swartz
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Biotechnology progress     Volume:  21     ISSN:  8756-7938     ISO Abbreviation:  Biotechnol. Prog.     Publication Date:    2005 Mar-Apr
Date Detail:
Created Date:  2005-04-01     Completed Date:  2005-07-06     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8506292     Medline TA:  Biotechnol Prog     Country:  United States    
Other Details:
Languages:  eng     Pagination:  460-5     Citation Subset:  IM    
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
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MeSH Terms
Cell-Free System
Electrophoresis, Polyacrylamide Gel
Escherichia coli / genetics*
Recombinant Proteins / biosynthesis*
Reg. No./Substance:
0/Recombinant Proteins

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