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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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SDS-PAGE of the expression and purification of PsGrx in E. coli. lane 1: IPTG-induced E. coli BL21 (Grx−); lane 2: a total cell lysate of IPTG-induced E. coli BL21 (Grx+); lane 3: purified PsGrx; lane 4: molecular weight protein marker.
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fig2: SDS-PAGE of the expression and purification of PsGrx in E. coli. lane 1: IPTG-induced E. coli BL21 (Grx−); lane 2: a total cell lysate of IPTG-induced E. coli BL21 (Grx+); lane 3: purified PsGrx; lane 4: molecular weight protein marker.

Mentions: The recombinant vector pETgrx was constructed and transformed into E. coli BL21(DE3). rPsGrx was overexpressed by IPTG induction, and SDS-PAGE results showed a strong band approximately 15.0 kDa was found compared to uninduced cells (Figure 2). The purification process resulted in a 5.81-fold purification with 43.83% final recovery and 53.25 U/mg specific activity.


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)

SDS-PAGE of the expression and purification of PsGrx in E. coli. lane 1: IPTG-induced E. coli BL21 (Grx−); lane 2: a total cell lysate of IPTG-induced E. coli BL21 (Grx+); lane 3: purified PsGrx; lane 4: molecular weight protein marker.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: SDS-PAGE of the expression and purification of PsGrx in E. coli. lane 1: IPTG-induced E. coli BL21 (Grx−); lane 2: a total cell lysate of IPTG-induced E. coli BL21 (Grx+); lane 3: purified PsGrx; lane 4: molecular weight protein marker.
Mentions: The recombinant vector pETgrx was constructed and transformed into E. coli BL21(DE3). rPsGrx was overexpressed by IPTG induction, and SDS-PAGE results showed a strong band approximately 15.0 kDa was found compared to uninduced cells (Figure 2). The purification process resulted in a 5.81-fold purification with 43.83% final recovery and 53.25 U/mg specific activity.

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