Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression

Jin Ho Ahn, Nam Young Kim, Tae Wan Kim, Jeong Mi Son, Taek Jin Kang, Chang Gil Park, Cha Yong Choi, Dong Myung Kim

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce 5-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.

Original languageEnglish
Pages (from-to)110-115
Number of pages6
JournalAnalytical Biochemistry
Volume345
Issue number1
DOIs
StatePublished - 1 Oct 2005

Keywords

  • ATP regeneration
  • Cell-free protein synthesis
  • Erythropoietin
  • In vitro protein synthesis
  • Nonsense suppression
  • Translation

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