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Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4364-9. doi: 10.1073/pnas.0712049105. Epub 2008 Mar 12.

Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae.

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1
Harvard Institute of Proteomics, Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber/Harvard Cancer Center DNA Resource Core, Harvard Medical School, 320 Charles Street, Cambridge, MA 02141, USA.

Abstract

Cholera, an infectious disease with global impact, is caused by pathogenic strains of the bacterium Vibrio cholerae. High-throughput functional proteomics technologies now offer the opportunity to investigate all aspects of the proteome, which has led to an increased demand for comprehensive protein expression clone resources. Genome-scale reagents for cholera would encourage comprehensive analyses of immune responses and systems-wide functional studies that could lead to improved vaccine and therapeutic strategies. Here, we report the production of the FLEXGene clone set for V. cholerae O1 biovar eltor str. N16961: a complete-genome collection of ORF clones. This collection includes 3,761 sequence-verified clones from 3,887 targeted ORFs (97%). The ORFs were captured in a recombinational cloning vector to facilitate high-throughput transfer of ORF inserts into suitable expression vectors. To demonstrate its application, approximately 15% of the collection was transferred into the relevant expression vector and used to produce a protein microarray by transcribing, translating, and capturing the proteins in situ on the array surface with 92% success. In a second application, a method to screen for protein triggers of Toll-like receptors (TLRs) was developed. We tested in vitro-synthesized proteins for their ability to stimulate TLR5 in A549 cells. This approach appropriately identified FlaC, and previously uncharacterized TLR5 agonist activities. These data suggest that the genome-scale, fully sequenced ORF collection reported here will be useful for high-throughput functional proteomic assays, immune response studies, structure biology, and other applications.

PMID:
18337508
PMCID:
PMC2393773
DOI:
10.1073/pnas.0712049105
[Indexed for MEDLINE]
Free PMC Article

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