Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis

C. Eric Hodgman, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7±0.5μgmL-1 with batch reactions lasting up to 2h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (μg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ~2×10-4 to ~4×10-1μg$-1, which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries.

Original languageEnglish (US)
Pages (from-to)2643-2654
Number of pages12
JournalBiotechnology and Bioengineering
Volume110
Issue number10
DOIs
StatePublished - Oct 2013

Keywords

  • Cell-free biology
  • Cell-free protein synthesis
  • In vitro translation
  • Protein expression
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Fingerprint

Dive into the research topics of 'Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis'. Together they form a unique fingerprint.

Cite this