Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display

• Published in: PloS one Vol. 3; no. 8; p. e2983
• Main Authors: Gao, Haichun, Pattison, Donna, Yan, Tingfen, Klingeman, Dawn M, Wang, Xiaohu, Petrosino, Joseph, Hemphill, Lisa, Wan, Xiufeng, Leaphart, Adam B, Weinstock, George M, Palzkill, Timothy, Zhou, Jizhong
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20-08-2008; Public Library of Science (PLoS)
• Subjects: Analysis; Annotations; Antibiotics; Antigens; Bacterial Proteins - genetics; BASIC BIOLOGICAL SCIENCES; Chromatography; Cloning; Cloning, Molecular; Cytochrome; Deoxyribonucleic acid; DNA; DNA microarrays; DNA polymerase; DNA, Bacterial - genetics; Drug resistance; E coli; Environmental science; Escherichia coli; Escherichia coli - genetics; Expression vectors; Gene Amplification; Gene expression; Gene Expression Regulation, Bacterial; Genes; Genetic research; Genetic Vectors; Genetics and Genomics; Genetics and Genomics/Functional Genomics; Genetics and Genomics/Genomics; Genome, Bacterial; Genomes; Genomics; In vivo methods and tests; Laboratories; Mass spectrometry; Mass spectroscopy; Medicine; Microbiology/Microbial Evolution and Genomics; NMR; Nuclear magnetic resonance; Open Reading Frames; Peptide Library; Phage display; Phages; Plasmids; Protein binding; Protein expression; Protein purification; Proteins; Proteomics; Recombinase; Regulation; Reproducibility of Results; Respiration; Science & Technology - Other Topics; Scientific imaging; Shewanella - genetics; Shewanella - virology; Thioredoxin; Treponema pallidum; United States > US; Tennessee; Oklahoma; Texas
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0002983

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST-tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro.