Enhanced Production of Soluble Recombinant Proteins With an In Situ-Removable Fusion Partner in a Cell-Free Synthesis System

Biotechnol J. 2017 Nov;12(11). doi: 10.1002/biot.201700125. Epub 2017 Sep 11.


High-yield production of soluble protein is a common concern in diverse fields of biotechnology. In this study, a strategy of using an engineered nucleotide sequence of ubiquitin for enhancing the production of soluble proteins in a cell-free synthesis system is presented. When examined for a series of proteins that otherwise were poorly expressed, N-terminal fusion with ubiquitin significantly increased both the expression levels and solubility of the translational products. The effect of ubiquitin fusion was also markedly augmented by engineering the nucleotide sequence of ubiquitin, leading to several fold enhancements in soluble production of target proteins. Recombinant proteins were produced with their native amino acid sequences through in situ removal of ubiquitin during cell-free synthesis reactions in the presence of a deubiquitinase. The presented strategy could be employed as a facile route to prepare soluble proteins required for various applications.

Keywords: cell-free protein synthesis; fusion partner; in situ cleavage; protein solubility; ubiquitin.

MeSH terms

  • Cell-Free System / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Protein Engineering / methods*
  • Protein Folding
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Solubility
  • Ubiquitin / chemistry
  • Ubiquitin / genetics
  • Ubiquitin / metabolism


  • Recombinant Fusion Proteins
  • Ubiquitin