Enhanced Yield of Recombinant Proteins with Site-Specifically Incorporated Unnatural Amino Acids Using a Cell-Free Expression System

Sviatlana Smolskaya, Zhiwen Jonathan Zhang, Lital Alfonta

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 μg/mL of translated protein (comprising 25 μg in 50 μL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.

Original languageEnglish
Article numbere68363
JournalPLoS ONE
Volume8
Issue number7
DOIs
StatePublished - 2 Jul 2013

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (all)
  • Agricultural and Biological Sciences (all)
  • General

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