title: Engineering and characterization of a superfolder green fluorescent protein.
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.
authors.affiliation: Bioscience Division, MS-M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Pédelacq
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Cabantous
authors.forename: Timothy
authors.initials: T
authors.lastname: Tran
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Terwilliger
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Waldo
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D041543:Pyrobaculum / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins