title: Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display.
abstract: 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.
authors: Institute for Environmental Genomics, University of Oklahoma, Norman, Oklahoma, United States of America.
authors: Haichun
authors: H
authors: Gao
authors: Donna
authors: D
authors: Pattison
authors: Tingfen
authors: T
authors: Yan
authors: Dawn M
authors: DM
authors: Klingeman
authors: Xiaohu
authors: X
authors: Wang
authors: Joseph
authors: J
authors: Petrosino
authors: Lisa
authors: L
authors: Hemphill
authors: Xiufeng
authors: X
authors: Wan
authors: Adam B
authors: AB
authors: Leaphart
authors: George M
authors: GM
authors: Weinstock
authors: Timothy
authors: T
authors: Palzkill
authors: Jizhong
authors: J
authors: Zhou
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D016680:Genome, Bacterial; D016366:Open Reading Frames; D019151:Peptide Library; D040901:Proteomics; D015203:Reproducibility of Results; D020592:Shewanella / Q000235:genetics* / Q000821:virology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D019151:Peptide Library