Experimental mapping of soluble protein domains using a hierarchical approach

Pedelacq, Jean-Denis; Nguyen, Hau B.; Cabantous, Stephanie; Mark, Brian L.; Listwan, Pawel; Bell, Carolyn; Friedland, Natasha; Lockard, Meghan; Faille, Alexandre; Mourey, Lionel; Terwilliger, Thomas C.; Waldo, Geoffrey S.

doi:10.1093/nar/gkr548

Title: Experimental mapping of soluble protein domains using a hierarchical approach

Journal Article · Tue Jul 19 00:00:00 EDT 2011 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkr548· OSTI ID:1625489

Pedelacq, Jean-Denis ^[1]; Nguyen, Hau B. ^[2]; Cabantous, Stephanie ^[3]; Mark, Brian L. ^[4]; Listwan, Pawel ^[2]; Bell, Carolyn ^[2]; Friedland, Natasha ^[2]; Lockard, Meghan ^[2]; Faille, Alexandre ^[1]; Mourey, Lionel ^[1]; Terwilliger, Thomas C. ^[2]; Waldo, Geoffrey S. ^[2]

National Centre for Scientific Research (CNRS), Toulouse (France). Institut de Pharmacologie et de Biologie Structurale (IPBS); Universite de Toulouse (France)
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
INSERM, Toulouse (France). Cancer Research Center of Toulouse; Universite de Toulouse (France); Inst. Claudius Regaud, Toulouse (France)
Univ. of Manitoba, Winnipeg, MB (Canada)

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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Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC52-06NA25396

OSTI ID:: 1625489

Journal Information:: Nucleic Acids Research, Vol. 39, Issue 18; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

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High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System Massemin, Amélie; Cabantous, Stéphanie; Waldo, Geoffrey S. Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-9624-7_15	book	January 2019
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