Rapid matrix-assisted refolding of histidine-tagged proteins

Chembiochem. 2009 Mar 23;10(5):869-76. doi: 10.1002/cbic.200800697.

Abstract

The formation of inclusion bodies (IBs)--amorphous aggregates of misfolded insoluble protein--during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His(6)-tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence--it allows refolding at up to 1000-fold higher protein concentration than folding in-solution.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Buffers
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Histidine / metabolism*
  • Inclusion Bodies / metabolism*
  • Protein Conformation
  • Protein Folding*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*

Substances

  • Buffers
  • Recombinant Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Histidine