Expression of Proteins Using the Third Domain of the Escherichia Coli Periplasmic-Protein TolA as a Fusion Partner

Protein Expr Purif. 2003 Mar;28(1):173-81. doi: 10.1016/s1046-5928(02)00681-2.

Abstract

The third domain of the periplasmic protein TolA from Escherichia coli (TolAIII) was used as a fusion partner in the expression of various proteins from bacteria and eukaryotes. TolAIII is small domain, expressed in high yields as a soluble protein in the cytoplasm of E. coli. Proteins were linked to the C-terminus of TolAIII by a short flexible linker containing sites for endopeptidases. Three different vectors were prepared, containing sites for enterokinase, thrombin or factor Xa. Fusion proteins also contain a His(6)-Ser(2) tag at their N-terminus for easier purification. Up to 90 mg fusion protein per liter bacterial culture was obtained using these vectors. Colicin N R-domain was expressed with this system as a fusion and processed further for functional studies. The yield of final pure R-domain was doubled as compared to the direct expression. The system may prove to be useful in the preparation of other peptides and proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics*
  • Gene Expression
  • Models, Molecular
  • Molecular Sequence Data
  • Periplasm / chemistry*
  • Periplasmic Proteins / chemistry
  • Periplasmic Proteins / genetics
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Escherichia coli Proteins
  • Periplasmic Proteins
  • Recombinant Fusion Proteins
  • tolA protein, E coli