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Expression and Localization of an Hsp70 Protein in the Microsporidian Encephalitozoon cuniculi.

Jolly CE, Leonard CA, Hayman JR - Int J Microbiol (2010)

Bottom Line: An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis.In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so.In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Box 70579, Johnson City, TN 37614, USA.

ABSTRACT
Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and mature E. cuniculi spores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.

No MeSH data available.


Related in: MedlinePlus

Heterologous expression of E. cuniculi C1 Hsp70 protein as a histidine fusion protein in E. coli and Western blot analysis using purified C1 antibodies.  A Coomassie stained SDS-PAGE gel ((a), right panel) of uninduced and IPTG induced expression of the recombinant protein shows a ~76 kDa protein in the induced lane.  A Western blot of the SDS-PAGE gel was performed using histidine-tag-specific antibodies ((a), left panel) confirmed the recombinant protein induction.   (b) A single ~76 kDa band was detected on 1D Western blot of E. cuniculi and E. intestinalis total spore protein using purified C1 Hsp70 protein antibodies.  (c) The C1 (ECU02_0100) and B1 (ECU03_0520) Hsp70-related proteins were identified from a Coomassie stained 2D SDS-PAGE gel of E. cuniculi total spore protein by MALDI-MS analysis of trypsin digested gel spots.  (d) Western analysis of the 2D gel using the C1 specific antibodies detected only the C1 protein (d).
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fig1: Heterologous expression of E. cuniculi C1 Hsp70 protein as a histidine fusion protein in E. coli and Western blot analysis using purified C1 antibodies. A Coomassie stained SDS-PAGE gel ((a), right panel) of uninduced and IPTG induced expression of the recombinant protein shows a ~76 kDa protein in the induced lane. A Western blot of the SDS-PAGE gel was performed using histidine-tag-specific antibodies ((a), left panel) confirmed the recombinant protein induction. (b) A single ~76 kDa band was detected on 1D Western blot of E. cuniculi and E. intestinalis total spore protein using purified C1 Hsp70 protein antibodies. (c) The C1 (ECU02_0100) and B1 (ECU03_0520) Hsp70-related proteins were identified from a Coomassie stained 2D SDS-PAGE gel of E. cuniculi total spore protein by MALDI-MS analysis of trypsin digested gel spots. (d) Western analysis of the 2D gel using the C1 specific antibodies detected only the C1 protein (d).

Mentions: The open reading frame that encodes from the N-terminal methionine residue to the C-terminal leucine residue immediately prior to the termination codon of C1 was amplified, cloned, and expressed in E. coli. The expressed recombinant protein was similar to the predicted size of 76.2 kDa (Figure 1(a)) and was used to immunize naïve rabbits. In one-dimensional Western blot analysis using purified spore protein from both E. intestinalis and E. cuniculi, the anti-C1 Hsp70 protein antibodies recognized a single protein band from both species (Figure 1(b)). However, our analysis of the E. cuniculi genome database identified a second Hsp70-related protein with a predicted size of 74.8 kDa (ECU03_0520; “B1”). Because the masses of these two proteins are so similar and the predicted amino acid identity is 28.3%, the single band observed in 1D Westerns may represent recognition of both the C1 and B1 Hsps. Such reactivity would be difficult to distinguish using one-dimensional SDS-PAGE. Therefore, two-dimensional SDS-PAGE electrophoresis and Western blotting analysis were performed with purified E. cuniculi spore proteins and the anti-C1 Hsp70 antibodies. Matrix-assisted laser desorption ionization mass spectrometry analysis of trypsin digested Coomassie gel spots identified both the C1 and B1 Hsp70 proteins (Figure 1(c)). Peptide coverage represented 38 and 24% of the C1 and B1 proteins, respectively. Interestingly, the B1 protein, which is predicted to be 74.8 kDa, is slightly larger in mass on the 2D SDS-PAGE gel than the predicted 76.2 kDa C1 protein. It is possible that the removal of the signal peptide from the C1 could account for the size discrepancy. Nonetheless, Western blotting of the two dimensional SDS-PAGE shows that the anti-C1 Hsp70 antibodies are specific for the C1 Hsp70-related protein and do not cross react with the similar sized B1 protein (Figure 1(d)).


Expression and Localization of an Hsp70 Protein in the Microsporidian Encephalitozoon cuniculi.

Jolly CE, Leonard CA, Hayman JR - Int J Microbiol (2010)

Heterologous expression of E. cuniculi C1 Hsp70 protein as a histidine fusion protein in E. coli and Western blot analysis using purified C1 antibodies.  A Coomassie stained SDS-PAGE gel ((a), right panel) of uninduced and IPTG induced expression of the recombinant protein shows a ~76 kDa protein in the induced lane.  A Western blot of the SDS-PAGE gel was performed using histidine-tag-specific antibodies ((a), left panel) confirmed the recombinant protein induction.   (b) A single ~76 kDa band was detected on 1D Western blot of E. cuniculi and E. intestinalis total spore protein using purified C1 Hsp70 protein antibodies.  (c) The C1 (ECU02_0100) and B1 (ECU03_0520) Hsp70-related proteins were identified from a Coomassie stained 2D SDS-PAGE gel of E. cuniculi total spore protein by MALDI-MS analysis of trypsin digested gel spots.  (d) Western analysis of the 2D gel using the C1 specific antibodies detected only the C1 protein (d).
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Related In: Results  -  Collection

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fig1: Heterologous expression of E. cuniculi C1 Hsp70 protein as a histidine fusion protein in E. coli and Western blot analysis using purified C1 antibodies. A Coomassie stained SDS-PAGE gel ((a), right panel) of uninduced and IPTG induced expression of the recombinant protein shows a ~76 kDa protein in the induced lane. A Western blot of the SDS-PAGE gel was performed using histidine-tag-specific antibodies ((a), left panel) confirmed the recombinant protein induction. (b) A single ~76 kDa band was detected on 1D Western blot of E. cuniculi and E. intestinalis total spore protein using purified C1 Hsp70 protein antibodies. (c) The C1 (ECU02_0100) and B1 (ECU03_0520) Hsp70-related proteins were identified from a Coomassie stained 2D SDS-PAGE gel of E. cuniculi total spore protein by MALDI-MS analysis of trypsin digested gel spots. (d) Western analysis of the 2D gel using the C1 specific antibodies detected only the C1 protein (d).
Mentions: The open reading frame that encodes from the N-terminal methionine residue to the C-terminal leucine residue immediately prior to the termination codon of C1 was amplified, cloned, and expressed in E. coli. The expressed recombinant protein was similar to the predicted size of 76.2 kDa (Figure 1(a)) and was used to immunize naïve rabbits. In one-dimensional Western blot analysis using purified spore protein from both E. intestinalis and E. cuniculi, the anti-C1 Hsp70 protein antibodies recognized a single protein band from both species (Figure 1(b)). However, our analysis of the E. cuniculi genome database identified a second Hsp70-related protein with a predicted size of 74.8 kDa (ECU03_0520; “B1”). Because the masses of these two proteins are so similar and the predicted amino acid identity is 28.3%, the single band observed in 1D Westerns may represent recognition of both the C1 and B1 Hsps. Such reactivity would be difficult to distinguish using one-dimensional SDS-PAGE. Therefore, two-dimensional SDS-PAGE electrophoresis and Western blotting analysis were performed with purified E. cuniculi spore proteins and the anti-C1 Hsp70 antibodies. Matrix-assisted laser desorption ionization mass spectrometry analysis of trypsin digested Coomassie gel spots identified both the C1 and B1 Hsp70 proteins (Figure 1(c)). Peptide coverage represented 38 and 24% of the C1 and B1 proteins, respectively. Interestingly, the B1 protein, which is predicted to be 74.8 kDa, is slightly larger in mass on the 2D SDS-PAGE gel than the predicted 76.2 kDa C1 protein. It is possible that the removal of the signal peptide from the C1 could account for the size discrepancy. Nonetheless, Western blotting of the two dimensional SDS-PAGE shows that the anti-C1 Hsp70 antibodies are specific for the C1 Hsp70-related protein and do not cross react with the similar sized B1 protein (Figure 1(d)).

Bottom Line: An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis.In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so.In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Box 70579, Johnson City, TN 37614, USA.

ABSTRACT
Microsporidia spore surface proteins are an important, under investigated aspect of spore/host cell attachment and infection. For comparison analysis of surface proteins, we required an antibody control specific for an intracellular protein. An endoplasmic reticulum-associated heat shock protein 70 family member (Hsp70; ECU02_0100; "C1") was chosen for further analysis. DNA encoding the C1 hsp70 was amplified, cloned and used to heterologously express the C1 Hsp70 protein, and specific antiserum was generated. Two-dimensional Western blotting analysis showed that the purified antibodies were monospecific. Immunoelectron microscopy of developing and mature E. cuniculi spores revealed that the protein localized to internal structures and not to the spore surface. In spore adherence inhibition assays, the anti-C1 antibodies did not inhibit spore adherence to host cell surfaces, whereas antibodies to a known surface adhesin (EnP1) did so. In future studies, the antibodies to the 'C1' Hsp70 will be used to delineate spore surface protein expression.

No MeSH data available.


Related in: MedlinePlus