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Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile

Paper ID Volume ID Publish Year Pages File Format Full-Text
25667 43589 2006 10 PDF Available
Title
Cell-free protein synthesis at high temperatures using the lysate of a hyperthermophile
Abstract

Systems for cell-free protein synthesis available today are usually based on the lysates of either Escherichia coli, wheat germ or rabbit reticulocyte, and protein synthesis reactions using these extracts are performed at moderate temperatures (20–40 °C). We report here the development of a novel system for cell-free protein synthesis that can be operated at high temperatures using a lysate of the hyperthermophilic archaeon, Thermococcus kodakaraensis. With the system, cell-free protein synthesis of ChiAΔ4, a derivative of T. kodakaraensis chitinase (ChiA), was observed within a temperature range of 40–80 °C, with an optimum at 65 °C. Corresponding chitinase activity was also detected in the reaction mixtures after cell-free protein synthesis, indicating that the synthesized ChiAΔ4 folded in a proper tertiary structure. The maximum concentration of active ChiAΔ4 synthesized was determined to be approximately 1.3 μg/mL. A time course experiment indicated that the amount of synthesized ChiAΔ4 saturated within 30 min at 65 °C, and energy depletion was suggested to be the main cause of this saturation. We further developed a system for transcription and translation-coupled protein synthesis at high temperatures using a combination of T. kodakaraensis lysate and thermostable T7 RNA polymerase.

Keywords
Cell-free protein synthesis; Thermococcus; Hyperthermophile; Archaea
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 126, Issue 2, 1 November 2006, Pages 186–195
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