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Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Cai Y, Yan W, Xu W, Yin Y, He Y, Wang H, Zhang X - Curr. Microbiol. (2013)

Bottom Line: The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB.Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.

ABSTRACT
Streptococcus pneumoniae DnaJ is recognized as a virulence factor whose role in pneumococcal virulence remains unclear. Here, we attempted to reveal the contribution of DnaJ in pneumococcal virulence from the identification of its interacting proteins using co-immunoprecipitation method. dnaJ was cloned into plasmid pAE03 generating pAE03-dnaJ-gfp which was used to transform S. pneumoniae D39 strain. Then anti-GFP coated beads were used to capture GFP-coupled proteins from the bacterial lysate. The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We finally obtained nine proteins such as DnaK, Gap, Eno, SpxB using this method. Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB. Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature. These results help to understand the role of DnaJ in the pathogenesis of S. pneumoniae.

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Differential DnaJ interaction proteins between S. pneumoniae strain D39 and D39-dnaJ-gfp by co-IP. S. pneumoniae strain D39 and D39-dnaJ-gfp were cultured in C+Y medium until OD600 0.4–0.5. Then the bacteria were collected and the pellet was sonicated. Cell debris was removed by centrifugation at 12,000 rpm for 30 min. The supernatant was collected and used for co-IP. Lanes 1 and 2 showed the proteins attached to protein G-agarose beads in S. pneumoniae D39-dnaJ-gfp or D39, respectively
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Fig2: Differential DnaJ interaction proteins between S. pneumoniae strain D39 and D39-dnaJ-gfp by co-IP. S. pneumoniae strain D39 and D39-dnaJ-gfp were cultured in C+Y medium until OD600 0.4–0.5. Then the bacteria were collected and the pellet was sonicated. Cell debris was removed by centrifugation at 12,000 rpm for 30 min. The supernatant was collected and used for co-IP. Lanes 1 and 2 showed the proteins attached to protein G-agarose beads in S. pneumoniae D39-dnaJ-gfp or D39, respectively

Mentions: The procedures of co-IP were mainly described as the protocol for GFP antibody with some modifications. As a result, we obtained 15 differential proteins attached on protein G-agarose beads between S. pneumoniae D39-dnaJ-gfp and WT D39 (Fig. 2).Fig. 2


Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Cai Y, Yan W, Xu W, Yin Y, He Y, Wang H, Zhang X - Curr. Microbiol. (2013)

Differential DnaJ interaction proteins between S. pneumoniae strain D39 and D39-dnaJ-gfp by co-IP. S. pneumoniae strain D39 and D39-dnaJ-gfp were cultured in C+Y medium until OD600 0.4–0.5. Then the bacteria were collected and the pellet was sonicated. Cell debris was removed by centrifugation at 12,000 rpm for 30 min. The supernatant was collected and used for co-IP. Lanes 1 and 2 showed the proteins attached to protein G-agarose beads in S. pneumoniae D39-dnaJ-gfp or D39, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824243&req=5

Fig2: Differential DnaJ interaction proteins between S. pneumoniae strain D39 and D39-dnaJ-gfp by co-IP. S. pneumoniae strain D39 and D39-dnaJ-gfp were cultured in C+Y medium until OD600 0.4–0.5. Then the bacteria were collected and the pellet was sonicated. Cell debris was removed by centrifugation at 12,000 rpm for 30 min. The supernatant was collected and used for co-IP. Lanes 1 and 2 showed the proteins attached to protein G-agarose beads in S. pneumoniae D39-dnaJ-gfp or D39, respectively
Mentions: The procedures of co-IP were mainly described as the protocol for GFP antibody with some modifications. As a result, we obtained 15 differential proteins attached on protein G-agarose beads between S. pneumoniae D39-dnaJ-gfp and WT D39 (Fig. 2).Fig. 2

Bottom Line: The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB.Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.

ABSTRACT
Streptococcus pneumoniae DnaJ is recognized as a virulence factor whose role in pneumococcal virulence remains unclear. Here, we attempted to reveal the contribution of DnaJ in pneumococcal virulence from the identification of its interacting proteins using co-immunoprecipitation method. dnaJ was cloned into plasmid pAE03 generating pAE03-dnaJ-gfp which was used to transform S. pneumoniae D39 strain. Then anti-GFP coated beads were used to capture GFP-coupled proteins from the bacterial lysate. The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We finally obtained nine proteins such as DnaK, Gap, Eno, SpxB using this method. Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB. Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature. These results help to understand the role of DnaJ in the pathogenesis of S. pneumoniae.

Show MeSH
Related in: MedlinePlus