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Total amino acid stabilization during cell-free protein synthesis reactions

Paper ID Volume ID Publish Year Pages File Format Full-Text
25652 43588 2006 11 PDF Available
Title
Total amino acid stabilization during cell-free protein synthesis reactions
Abstract

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75–250% relative to cell-free expression using the control extract.

Keywords
Cell-free biology; Cell-free protein synthesis; Amino acid stabilization; Escherichia coli; Cell extract
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 123, Issue 2, 17 May 2006, Pages 193–203
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us