Generation and validation of a Shewanella oneidensis MR-1 clone set for protein expression and phage display.
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.
Gao
Haichun
H
Institute for Environmental Genomics, University of Oklahoma, Norman, Oklahoma, United States of America.
Pattison
Donna
D
Yan
Tingfen
T
Klingeman
Dawn M
DM
Wang
Xiaohu
X
Petrosino
Joseph
J
Hemphill
Lisa
L
Wan
Xiufeng
X
Leaphart
Adam B
AB
Weinstock
George M
GM
Palzkill
Timothy
T
Zhou
Jizhong
J
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
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D019151:Peptide Library