title: Experimental mapping of soluble protein domains using a hierarchical approach.
abstract: Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.
authors.affiliation: CNRS; IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, F-31077 Toulouse, France. Jean-Denis.Pedelacq@ipbs.fr
authors.forename: Jean-Denis
authors.initials: JD
authors.lastname: Pedelacq
authors.forename: Hau B
authors.initials: HB
authors.lastname: Nguyen
authors.forename: Stephanie
authors.initials: S
authors.lastname: Cabantous
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
authors.forename: Pawel
authors.initials: P
authors.lastname: Listwan
authors.forename: Carolyn
authors.initials: C
authors.lastname: Bell
authors.forename: Natasha
authors.initials: N
authors.lastname: Friedland
authors.forename: Meghan
authors.initials: M
authors.lastname: Lockard
authors.forename: Alexandre
authors.initials: A
authors.lastname: Faille
authors.forename: Lionel
authors.initials: L
authors.lastname: Mourey
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Terwilliger
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Waldo
mesh_terms: D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D012995:Solubility; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D049452:Green Fluorescent Proteins; D048630:Polyketide Synthases; D013762:Tetrahydrofolate Dehydrogenase; D058543:Class Ia Phosphatidylinositol 3-Kinase