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Expression, purification, and functional analysis of novel RelE operon from X. nematophila.

Rathore JS, Gautam LK - ScientificWorldJournal (2014)

Bottom Line: Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified.This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 303 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA ; School of Biotechnology, Gautam Buddha University, Greater Noida, Yamuna Expressway, Uttar Pradesh 201312, India.

ABSTRACT
Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

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Related in: MedlinePlus

Expression profile of recombinant Xn-relE toxin protein. SDS-PAGE showing expression of Xn-relE toxin gene. (a) Lane M, protein marker; lane 1, induced cells harbouring pGEX4T1 vector; lane 2, induced cells from clone 1. (b) Purification of recombinant GST tagged Xn-relE protein by GST affinity chromatography. Lanes 1, 2, and 3, purified recombinant GST tagged Xn-relE protein.
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fig2: Expression profile of recombinant Xn-relE toxin protein. SDS-PAGE showing expression of Xn-relE toxin gene. (a) Lane M, protein marker; lane 1, induced cells harbouring pGEX4T1 vector; lane 2, induced cells from clone 1. (b) Purification of recombinant GST tagged Xn-relE protein by GST affinity chromatography. Lanes 1, 2, and 3, purified recombinant GST tagged Xn-relE protein.

Mentions: Toxin-antitoxin modules are mainly categorized in three different classes and class II TA system comprises proteins forming toxin-antitoxin (TA) protein complex [3, 21]. Xn-relE/EAT proteins from X. nematophila have shown similarity with proteins from Rel family and hence are supposed to form protein complex. Genomic organization of the novel Xn-relE/EAT operon is shown in Figure 1 indicating overlapping 11 base pairs in between. In this study an attempt was made for the expression of Xn-relE gene in pET-28 vector but results were not satisfactory. Then Xn-relE toxin gene alone was cloned in pGEX4T1 expression vector and expression profile is shown in Figure 2(a). Band as shown with arrow in lane 2 was visible in SDS-PAGE at the position above 34 kDa protein marker which is corroborated with the size of GST fusion with Xn-relE recombinant protein. Corresponding band was missing in the induced cells containing empty vector as shown in lane 1 in Figure 2(a). GST tagged recombinant Xn-relE protein was purified with GST affinity chromatography under optimal conditions. Single band was visible in SDS-PAGE at the position below 34 kDa protein marker which is corroborated with the size of GST fusion with Xn-relE recombinant protein as shown in Figure 2(b). From the study it has been confirmed that Xn-relE gene is encoded for a novel toxic protein whose nature and degree of endotoxicity were also studied in this study which signifies its functional similarities with other toxins from Rel family [22–24].


Expression, purification, and functional analysis of novel RelE operon from X. nematophila.

Rathore JS, Gautam LK - ScientificWorldJournal (2014)

Expression profile of recombinant Xn-relE toxin protein. SDS-PAGE showing expression of Xn-relE toxin gene. (a) Lane M, protein marker; lane 1, induced cells harbouring pGEX4T1 vector; lane 2, induced cells from clone 1. (b) Purification of recombinant GST tagged Xn-relE protein by GST affinity chromatography. Lanes 1, 2, and 3, purified recombinant GST tagged Xn-relE protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4265723&req=5

fig2: Expression profile of recombinant Xn-relE toxin protein. SDS-PAGE showing expression of Xn-relE toxin gene. (a) Lane M, protein marker; lane 1, induced cells harbouring pGEX4T1 vector; lane 2, induced cells from clone 1. (b) Purification of recombinant GST tagged Xn-relE protein by GST affinity chromatography. Lanes 1, 2, and 3, purified recombinant GST tagged Xn-relE protein.
Mentions: Toxin-antitoxin modules are mainly categorized in three different classes and class II TA system comprises proteins forming toxin-antitoxin (TA) protein complex [3, 21]. Xn-relE/EAT proteins from X. nematophila have shown similarity with proteins from Rel family and hence are supposed to form protein complex. Genomic organization of the novel Xn-relE/EAT operon is shown in Figure 1 indicating overlapping 11 base pairs in between. In this study an attempt was made for the expression of Xn-relE gene in pET-28 vector but results were not satisfactory. Then Xn-relE toxin gene alone was cloned in pGEX4T1 expression vector and expression profile is shown in Figure 2(a). Band as shown with arrow in lane 2 was visible in SDS-PAGE at the position above 34 kDa protein marker which is corroborated with the size of GST fusion with Xn-relE recombinant protein. Corresponding band was missing in the induced cells containing empty vector as shown in lane 1 in Figure 2(a). GST tagged recombinant Xn-relE protein was purified with GST affinity chromatography under optimal conditions. Single band was visible in SDS-PAGE at the position below 34 kDa protein marker which is corroborated with the size of GST fusion with Xn-relE recombinant protein as shown in Figure 2(b). From the study it has been confirmed that Xn-relE gene is encoded for a novel toxic protein whose nature and degree of endotoxicity were also studied in this study which signifies its functional similarities with other toxins from Rel family [22–24].

Bottom Line: Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified.This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 303 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA ; School of Biotechnology, Gautam Buddha University, Greater Noida, Yamuna Expressway, Uttar Pradesh 201312, India.

ABSTRACT
Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms. Escherichia coli RelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin from Xenorhabdus nematophila possessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter in E. coli Top 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

Show MeSH
Related in: MedlinePlus