Nature Biotechnology 24, 79 - 88 (2005)
Published online: 20 December 2005; | doi:10.1038/nbt1172
There is a Corrigendum (September 2006) associated with this Article.Engineering and characterization of a superfolder green fluorescent proteinJean-Denis Pédelacq1, Stéphanie Cabantous1, Timothy Tran2, Thomas C Terwilliger1
& Geoffrey S Waldo11
Bioscience Division, MS-M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA. 2
Cornell University, Department of Chemistry & Chemical Biology, P.O. Box 305, Ithaca, New York 14851-0305, USA.
Correspondence should be addressed to Geoffrey S Waldo waldo@lanl.gov 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.
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