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.

A Crp-Dependent Two-Component System Regulates Nitrate and Nitrite Respiration in Shewanella oneidensis

“…To test whether these proteins function together in regulation of nitrate/nitrite respiration, we performed the trans-phosphorylation assay. S. oneidensis NarP, SO1860, and NarQ 51–585 were cloned into Gateway entry vector pDNOR221, transferred into a protein expression system to attach an N-terminal His-tag, and the His-tagged proteins were expressed in E. coli and purified, mostly from inclusion bodies as described previously [37]. Like SO1860 [36], NarP failed to phosphorylate itself when ATP was included (Fig.…”

“…To determine this and whether specific phosphorylation is required for NarP DNA binding, a S. oneidensis NarP mutation protein in which Asp 57 was replaced with asparagine (57 DN ) by site-directed mutagenesis was created, expressed and purified from E. coli . The binding characteristics of the NarP, NarP-P, NarP(D57N), and NarP(D57N) # (treated by phosphorylation agents) proteins were tested using a radio-labeled nrfA promoter DNA probe, which has been shown to be able to bind to NarP [37].…”

Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments

“…Expression and purification of S. oneidensis Crp protein and EMSA The entire clone set of S. oneidensis open reading frames has been constructed, as reported previously (Gao et al, 2008b). The crp gene within pDONR221 (the entry vector) was transferred to pTP247 (the destination His-tag expression vector).…”

“…The crp gene within pDONR221 (the entry vector) was transferred to pTP247 (the destination His-tag expression vector). Protein expression and purification was performed, as previously described (Gao et al, 2008b). The probes used for electrophoretic motility shift assay (EMSA) were prepared by PCR with 33 P end-labeled primers.…”

“…The His-tagged Crp protein was produced in E. coli and purified from inclusion bodies (Gao et al, 2008b). It has been previously shown that expression of the dms operon (encoding dimethyl sulfoxide reductase) is dependent on Crp.…”