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Article Dans Une Revue Nucleic Acids Research Année : 2011

Experimental mapping of soluble protein domains using a hierarchical approach

Hau B Nguyen
  • Fonction : Auteur
Stephanie Cabantous
Brian L Mark
  • Fonction : Auteur
Pawel Listwan
  • Fonction : Auteur
Carolyn Bell
  • Fonction : Auteur
Natasha Friedland
  • Fonction : Auteur
Meghan Lockard
  • Fonction : Auteur
Thomas C Terwilliger
  • Fonction : Auteur
Geoffrey S Waldo
  • Fonction : Auteur

Résumé

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 p85a 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.
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Dates et versions

hal-03003363 , version 1 (13-11-2020)

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Jean-Denis Pedelacq, Hau B Nguyen, Stephanie Cabantous, Brian L Mark, Pawel Listwan, et al.. Experimental mapping of soluble protein domains using a hierarchical approach. Nucleic Acids Research, 2011, 39, pp.e125 - e125. ⟨10.1093/nar/gkr548⟩. ⟨hal-03003363⟩
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