title: Recent advances in GFP folding reporter and split-GFP solubility reporter technologies. Application to improving the folding and solubility of recalcitrant proteins from Mycobacterium tuberculosis.
abstract: We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691-695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102-107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, MS-M888, Los Alamos, NM 87545, USA.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Cabantous
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Pédelacq
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
authors.forename: Cleo
authors.initials: C
authors.lastname: Naranjo
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 / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins