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Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

Zhang Y, Bae YA, Zong HY, Kong Y, Cai GB - Iran J Parasitol (2016 Jan-Mar)

Bottom Line: After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed.The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates.TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Genetics, Wuhan University School of Basic Medicial Sciences, Wuhan 430071, China.

ABSTRACT

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.

Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.

Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.

Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE, western blot and zymograph analysis of recombinant mTsMP enzyme. (A) Expressed protein were analyzed by 12% SDS-PAGE stained with Coomassie blue. Lane 1 and lane 3, soluble and insoluble fraction from Escherichia coli cell lysates which did not induce with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG), respectively; lane 2 and lane 4, soluble and insoluble fraction from E. coli cell lysates which induced with 0.5 mM IPTG, respectively; lane 5, purified recombinant mTsMP protein by Glutathione Sepharoase 4B affinity chromatography and ion-exchange chromatography with Q-Sepharose beads. The molecular weight marker in kilodaltons (kDa) was shown at the left. (B) Western blot analysis for purified recombinant mTsMP with anti-GST antibody as primary antibody (lane 6); (C) The activated recombinant TsMP was analyzed by substrate gel zymography with 0.2% gelatin as a substrate (lane 7)
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Figure 2: SDS-PAGE, western blot and zymograph analysis of recombinant mTsMP enzyme. (A) Expressed protein were analyzed by 12% SDS-PAGE stained with Coomassie blue. Lane 1 and lane 3, soluble and insoluble fraction from Escherichia coli cell lysates which did not induce with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG), respectively; lane 2 and lane 4, soluble and insoluble fraction from E. coli cell lysates which induced with 0.5 mM IPTG, respectively; lane 5, purified recombinant mTsMP protein by Glutathione Sepharoase 4B affinity chromatography and ion-exchange chromatography with Q-Sepharose beads. The molecular weight marker in kilodaltons (kDa) was shown at the left. (B) Western blot analysis for purified recombinant mTsMP with anti-GST antibody as primary antibody (lane 6); (C) The activated recombinant TsMP was analyzed by substrate gel zymography with 0.2% gelatin as a substrate (lane 7)

Mentions: A mature catalytic domain of TsMP was amplified, cloned into the pGEX-6P-1 prokaryotic expression vector and transformed into E. coli BL21 (DE3). The recombinant mTsMP protein was expressed in E. coli as an insoluble protein with an apparent molecular mass of 52 kDa (Fig. 2A), which is consistent with the estimated molecular mass of GST (26 kDa) and the deduced amino acid sequence for mTsMP (26 kDa). After dialysis and refolding, the recombinant protein was purified by Glutathione Sepharoase 4B affinity chromatography and further purified by ion-exchange chromatography with Q-Sepharose beads. Western blotting result with anti-GST antibody as primary antibody demonstrated that the recombinant protein was the GST-fusion protein (Fig. 2B). Protease activity of the refolded recombinant GST-mTsMP was confirmed by substrate gel zymograph (Fig. 2C).


Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

Zhang Y, Bae YA, Zong HY, Kong Y, Cai GB - Iran J Parasitol (2016 Jan-Mar)

SDS-PAGE, western blot and zymograph analysis of recombinant mTsMP enzyme. (A) Expressed protein were analyzed by 12% SDS-PAGE stained with Coomassie blue. Lane 1 and lane 3, soluble and insoluble fraction from Escherichia coli cell lysates which did not induce with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG), respectively; lane 2 and lane 4, soluble and insoluble fraction from E. coli cell lysates which induced with 0.5 mM IPTG, respectively; lane 5, purified recombinant mTsMP protein by Glutathione Sepharoase 4B affinity chromatography and ion-exchange chromatography with Q-Sepharose beads. The molecular weight marker in kilodaltons (kDa) was shown at the left. (B) Western blot analysis for purified recombinant mTsMP with anti-GST antibody as primary antibody (lane 6); (C) The activated recombinant TsMP was analyzed by substrate gel zymography with 0.2% gelatin as a substrate (lane 7)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4835468&req=5

Figure 2: SDS-PAGE, western blot and zymograph analysis of recombinant mTsMP enzyme. (A) Expressed protein were analyzed by 12% SDS-PAGE stained with Coomassie blue. Lane 1 and lane 3, soluble and insoluble fraction from Escherichia coli cell lysates which did not induce with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG), respectively; lane 2 and lane 4, soluble and insoluble fraction from E. coli cell lysates which induced with 0.5 mM IPTG, respectively; lane 5, purified recombinant mTsMP protein by Glutathione Sepharoase 4B affinity chromatography and ion-exchange chromatography with Q-Sepharose beads. The molecular weight marker in kilodaltons (kDa) was shown at the left. (B) Western blot analysis for purified recombinant mTsMP with anti-GST antibody as primary antibody (lane 6); (C) The activated recombinant TsMP was analyzed by substrate gel zymography with 0.2% gelatin as a substrate (lane 7)
Mentions: A mature catalytic domain of TsMP was amplified, cloned into the pGEX-6P-1 prokaryotic expression vector and transformed into E. coli BL21 (DE3). The recombinant mTsMP protein was expressed in E. coli as an insoluble protein with an apparent molecular mass of 52 kDa (Fig. 2A), which is consistent with the estimated molecular mass of GST (26 kDa) and the deduced amino acid sequence for mTsMP (26 kDa). After dialysis and refolding, the recombinant protein was purified by Glutathione Sepharoase 4B affinity chromatography and further purified by ion-exchange chromatography with Q-Sepharose beads. Western blotting result with anti-GST antibody as primary antibody demonstrated that the recombinant protein was the GST-fusion protein (Fig. 2B). Protease activity of the refolded recombinant GST-mTsMP was confirmed by substrate gel zymograph (Fig. 2C).

Bottom Line: After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed.The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates.TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Genetics, Wuhan University School of Basic Medicial Sciences, Wuhan 430071, China.

ABSTRACT

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.

Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.

Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.

Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

No MeSH data available.


Related in: MedlinePlus