Engineering and Characterization of a Superfolder Green Fluorescent Protein

Nat Biotechnol. 2006 Jan;24(1):79-88. doi: 10.1038/nbt1172. Epub 2005 Dec 20.

Abstract

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / analysis
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Models, Molecular
  • Molecular Conformation
  • Protein Binding
  • Protein Engineering / methods*
  • Protein Folding
  • Pyrobaculum / genetics
  • Pyrobaculum / metabolism*
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Fluorescence / methods

Substances

  • Bacterial Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins

Associated data

  • PDB/2B3P
  • PDB/2B3Q