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

Gao H, Pattison D, Yan T, Klingeman DM, Wang X, Petrosino J, Hemphill L, Wan X, Leaphart AB, Weinstock GM, Palzkill T, Zhou J - PLoS ONE (2008)

Bottom Line: Success rates for heterologous protein expression of S. oneidensis His- or His/GST-tagged proteins in E. coli were approximately 70%.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Environmental Genomics, University of Oklahoma, Norman, Oklahoma, United States of America.

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.

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SDS-PAGE of S. oneidensis NarP expressed from the destination vector pTP247.A. Lane 1: protein marker; Lane 2: Soluble protein after lysis of E. coli expressing NarP protein. B. Electrophoretic gel shift assays of NarP and phosphorylated NarP-P. Increasing concentrations (0, 0.65, 1.3, 2.6, and 5 µM) of unphosphorylated NarP and phosphorylated NarP are shown. DNA is stained with CyberGreen. The 100bp ladder was used in the experiments.
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pone-0002983-g002: SDS-PAGE of S. oneidensis NarP expressed from the destination vector pTP247.A. Lane 1: protein marker; Lane 2: Soluble protein after lysis of E. coli expressing NarP protein. B. Electrophoretic gel shift assays of NarP and phosphorylated NarP-P. Increasing concentrations (0, 0.65, 1.3, 2.6, and 5 µM) of unphosphorylated NarP and phosphorylated NarP are shown. DNA is stained with CyberGreen. The 100bp ladder was used in the experiments.

Mentions: Protein expression and purification studies were pursued further for two of the response regulatory proteins, ArcA and NarP. As shown previously by Gao et al. [33], the purified ArcA protein can bind specifically to promoter DNA when phosphorylated/activated using carbamoyl phosphate according to electrophoretic gel mobility shift assays [33]. The non-phosphorylated protein did not show any specific DNA binding activity [33]. SO3982, showing sequence homology to E. coli NarP (72% similarity), was named as NarP in the latest annotation. NarP is a response regulator belonging to the LuxR family. Evidence from work in E. coli suggests that NarP may regulate its own expression [34], as well as expression of NrfA, a formate-dependent nitrite reductase enzyme [35]. In S. oneidensis, NarP and NrfA share the same promoter region, although the genes are transcribed in opposite directions. This promoter region was amplified via PCR and used in electrophoretic mobility shift assays. The cloned narP gene in the pDONR221 vector was transferred to the pTP247 His-tag destination vector and the NarP protein was expressed and purified in soluble form using metal affinity chromatography (Fig. 2A). The purified protein was phosphorylated with carbamoyl phosphate and electrophoretic mobility shift (EMSA) DNA binding studies indicated the phosphorylated SO3982 protein bound to a PCR product containing narP-nrfA promoter DNA while nonphosphorylated SO3982 protein did not (Fig. 2B). These results indicate that NarP phosphorylation is required for DNA binding. These studies demonstrate that the clone set and the pTP247 destination plasmid can be used to successfully express and purify proteins in E. coli.


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

Gao H, Pattison D, Yan T, Klingeman DM, Wang X, Petrosino J, Hemphill L, Wan X, Leaphart AB, Weinstock GM, Palzkill T, Zhou J - PLoS ONE (2008)

SDS-PAGE of S. oneidensis NarP expressed from the destination vector pTP247.A. Lane 1: protein marker; Lane 2: Soluble protein after lysis of E. coli expressing NarP protein. B. Electrophoretic gel shift assays of NarP and phosphorylated NarP-P. Increasing concentrations (0, 0.65, 1.3, 2.6, and 5 µM) of unphosphorylated NarP and phosphorylated NarP are shown. DNA is stained with CyberGreen. The 100bp ladder was used in the experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2500165&req=5

pone-0002983-g002: SDS-PAGE of S. oneidensis NarP expressed from the destination vector pTP247.A. Lane 1: protein marker; Lane 2: Soluble protein after lysis of E. coli expressing NarP protein. B. Electrophoretic gel shift assays of NarP and phosphorylated NarP-P. Increasing concentrations (0, 0.65, 1.3, 2.6, and 5 µM) of unphosphorylated NarP and phosphorylated NarP are shown. DNA is stained with CyberGreen. The 100bp ladder was used in the experiments.
Mentions: Protein expression and purification studies were pursued further for two of the response regulatory proteins, ArcA and NarP. As shown previously by Gao et al. [33], the purified ArcA protein can bind specifically to promoter DNA when phosphorylated/activated using carbamoyl phosphate according to electrophoretic gel mobility shift assays [33]. The non-phosphorylated protein did not show any specific DNA binding activity [33]. SO3982, showing sequence homology to E. coli NarP (72% similarity), was named as NarP in the latest annotation. NarP is a response regulator belonging to the LuxR family. Evidence from work in E. coli suggests that NarP may regulate its own expression [34], as well as expression of NrfA, a formate-dependent nitrite reductase enzyme [35]. In S. oneidensis, NarP and NrfA share the same promoter region, although the genes are transcribed in opposite directions. This promoter region was amplified via PCR and used in electrophoretic mobility shift assays. The cloned narP gene in the pDONR221 vector was transferred to the pTP247 His-tag destination vector and the NarP protein was expressed and purified in soluble form using metal affinity chromatography (Fig. 2A). The purified protein was phosphorylated with carbamoyl phosphate and electrophoretic mobility shift (EMSA) DNA binding studies indicated the phosphorylated SO3982 protein bound to a PCR product containing narP-nrfA promoter DNA while nonphosphorylated SO3982 protein did not (Fig. 2B). These results indicate that NarP phosphorylation is required for DNA binding. These studies demonstrate that the clone set and the pTP247 destination plasmid can be used to successfully express and purify proteins in E. coli.

Bottom Line: Success rates for heterologous protein expression of S. oneidensis His- or His/GST-tagged proteins in E. coli were approximately 70%.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Environmental Genomics, University of Oklahoma, Norman, Oklahoma, United States of America.

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.

Show MeSH
Related in: MedlinePlus