ArticleNucleic acids research2008
Identification of soluble protein fragments by gene fragmentation and genetic selection.
Article in Nucleic acids research, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 37 citations in OpenAlex.
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- An efficient ORF selection system for DNA fragment libraries based on split beta-lactamase complementation.PloS one · 2020Article
- Capturing the 'ome': the expanding molecular toolbox for RNA and DNA library construction.Nucleic acids research · 2018Review
- Directed evolution to improve protein folding in vivo.Current opinion in structural biology · 2018Review
- [Construction of the pIRES2-ZsGreen1 eukaryotic expression vector of Factor Ⅸ gene and expression in HEK-293 cells].Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi · 2016Article
- Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication.The Journal of biological chemistry · 2016Article
- Solution structure of a soluble fragment derived from a membrane protein by shotgun proteolysis.Protein engineering, design & selection : PEDS · 2015Article
- Library methods for structural biology of challenging proteins and their complexes.Current opinion in structural biology · 2013Review
- Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells.The Korean journal of chemical engineering · 2012Article
- Experimental mapping of soluble protein domains using a hierarchical approach.Nucleic acids research · 2011Article
- A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.Protein engineering, design & selection : PEDS · 2011Article
- CoESPRIT: a library-based construct screening method for identification and expression of soluble protein complexes.PloS one · 2011Article
- Mining mammalian genomes for folding competent proteins using Tat-dependent genetic selection in Escherichia coli.Protein science : a publication of the Protein Society · 2009Article
- Random dissection to select for protein split sites and its application in protein fragment complementation.Protein science : a publication of the Protein Society · 2009Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
Abstract
We describe a new method, which identifies protein fragments for soluble expression in Escherichia coli from a randomly fragmented gene library. Inhibition of E. coli dihydrofolate reductase (DHFR) by trimethoprim (TMP) prevents growth, but this can be relieved by murine DHFR (mDHFR). Bacterial strains expressing mDHFR fusions with the soluble proteins green fluroscent protein (GFP) or EphB2 (SAM domain) displayed markedly increased growth rates with TMP compared to strains expressing insoluble EphB2 (TK domain) or ketosteroid isomerase (KSI). Therefore, mDHFR is affected by the solubility of fusion partners and can act as a reporter of soluble protein expression. Random fragment libraries of the transcription factor Fli1 were generated by deoxyuridine incorporation and endonuclease V cleavage. The fragments were cloned upstream of mDHFR and TMP resistant clones expressing soluble protein were identified. These were found to cluster around the DNA binding ETS domain. A selected Fli1 fragment was expressed independently of mDHFR and was judged to be correctly folded by various biophysical methods including NMR. Soluble fragments of the cell-surface receptor Pecam1 were also identified. This genetic selection method was shown to generate expression clones useful for both structural studies and antibody generation and does not require a priori knowledge of domain architecture.
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