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Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.

Wang Q, Hou Y, Shi Y, Han X, Chen Q, Hu Z, Liu Y, Li Y - Biomed Res Int (2014)

Bottom Line: Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide.Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC.It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl.

View Article: PubMed Central - PubMed

Affiliation: School of Marine and Technology, Harbin Institute of Technology, Weihai 264209, China.

ABSTRACT
Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

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Biochemical properties of the rPsGrx. (a) Effect of temperatures on activity of the rPsGrx. (b) Effect of temperatures on the stability of the rPsGrx. The protein was incubated at 40°C closed triangles and 50°C closed circles for 10, 20, 30, and 40 min, respectively and then directly put into an ice water bath and the residual activity was measured in the standard assay. (c). Effect of pH on activity of the rPsGrx.
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fig3: Biochemical properties of the rPsGrx. (a) Effect of temperatures on activity of the rPsGrx. (b) Effect of temperatures on the stability of the rPsGrx. The protein was incubated at 40°C closed triangles and 50°C closed circles for 10, 20, 30, and 40 min, respectively and then directly put into an ice water bath and the residual activity was measured in the standard assay. (c). Effect of pH on activity of the rPsGrx.

Mentions: Effects of temperatures on the rPsGrx activity were measured in temperature range of 0°C–60°C (Figure 3(a)). The optimal temperature for rPsGrx activity was 30°C. rPsGrx showed about 47.2% and 25.5% of its activity at 10°C and 0°C, respectively. As for the thermal stability assay, rPsGrx was evaluated by incubation at temperature 40°C and 50°C for 40 min (Figure 3(b)). rPsGrx showed 65.0% of its activity after incubation at 40°C for 20 min while lost almost all the activity after incubation at 50°C for 30 min. Effect of pH on the Grx activity was studied using different pH buffers. As shown in Figure 3(c), rPsGrx showed activity in a broad pH range of 5.0–9.0. The maximal activity was observed at pH 8.0. But it was completely inactive in buffer pH 10.0.


Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.

Wang Q, Hou Y, Shi Y, Han X, Chen Q, Hu Z, Liu Y, Li Y - Biomed Res Int (2014)

Biochemical properties of the rPsGrx. (a) Effect of temperatures on activity of the rPsGrx. (b) Effect of temperatures on the stability of the rPsGrx. The protein was incubated at 40°C closed triangles and 50°C closed circles for 10, 20, 30, and 40 min, respectively and then directly put into an ice water bath and the residual activity was measured in the standard assay. (c). Effect of pH on activity of the rPsGrx.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109671&req=5

fig3: Biochemical properties of the rPsGrx. (a) Effect of temperatures on activity of the rPsGrx. (b) Effect of temperatures on the stability of the rPsGrx. The protein was incubated at 40°C closed triangles and 50°C closed circles for 10, 20, 30, and 40 min, respectively and then directly put into an ice water bath and the residual activity was measured in the standard assay. (c). Effect of pH on activity of the rPsGrx.
Mentions: Effects of temperatures on the rPsGrx activity were measured in temperature range of 0°C–60°C (Figure 3(a)). The optimal temperature for rPsGrx activity was 30°C. rPsGrx showed about 47.2% and 25.5% of its activity at 10°C and 0°C, respectively. As for the thermal stability assay, rPsGrx was evaluated by incubation at temperature 40°C and 50°C for 40 min (Figure 3(b)). rPsGrx showed 65.0% of its activity after incubation at 40°C for 20 min while lost almost all the activity after incubation at 50°C for 30 min. Effect of pH on the Grx activity was studied using different pH buffers. As shown in Figure 3(c), rPsGrx showed activity in a broad pH range of 5.0–9.0. The maximal activity was observed at pH 8.0. But it was completely inactive in buffer pH 10.0.

Bottom Line: Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide.Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC.It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl.

View Article: PubMed Central - PubMed

Affiliation: School of Marine and Technology, Harbin Institute of Technology, Weihai 264209, China.

ABSTRACT
Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.

Show MeSH
Related in: MedlinePlus