ArticleNucleic acids research2011
Experimental mapping of soluble protein domains using a hierarchical approach.
Article in Nucleic acids research, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein-protein interactions.Scientific reports · 2025Article
- Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis.BMC biology · 2022Article
- Massively parallel interrogation of protein fragment secretability using SECRiFY reveals features influencing secretory system transit.Nature communications · 2021Article
- An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation.PloS one · 2020Article
- Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology.International journal of molecular sciences · 2019Review
- The Uncommon Enzymology of Cis-Acyltransferase Assembly Lines.Chemical reviews · 2017Review
- Mass spectral determination of phosphopantetheinylation specificity for carrier proteins inFEBS open bio · 2016Article
- Solution structure of a soluble fragment derived from a membrane protein by shotgun proteolysis.Protein engineering, design & selection : PEDS · 2015Article
- Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.Proteins · 2015Article
- From deep sequencing to actual clones.Protein engineering, design & selection : PEDS · 2014Article
- A new protein-protein interaction sensor based on tripartite split-GFP association.Scientific reports · 2013Article
- Library methods for structural biology of challenging proteins and their complexes.Current opinion in structural biology · 2013Review
- A chemical proteomics approach to profiling the ATP-binding proteome of Mycobacterium tuberculosis.Molecular & cellular proteomics : MCP · 2013Article
- 4'-Phosphopantetheinyl transferase PptT, a new drug target required for Mycobacterium tuberculosis growth and persistence in vivo.PLoS pathogens · 2012Article
- Divergence of multimodular polyketide synthases revealed by a didomain structure.Nature chemical biology · 2012Article
Corrections and comments
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Authors and funding
12 authors.
Funding
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.
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Registered trials
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