Limits...
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.

Show MeSH

Related in: MedlinePlus

Identification of pAE03-dnaJ-gfp in S. pneumoniae strain D39-dnaJ-gfp. Two positive colonies (D39-dnaJ-gfp) were selected on BA plates supplemented with 0.25 μg/ml erythromycin, and confirmed by PCR with gfp primers (a). The samples were D39 (1), plasmid pAE03 (2), D39-dnaJ-gfp 2 (3), D39-dnaJ-gfp 1 (4) and marker (M).Then they were cultured in C+Y medium until OD600 0.4–0.5. Bacteria was collected by centrifugation at 12,000 rpm and washed twice with PBS. The pellet was then resuspended in 50–80 μl 2× SDS sample buffer. Samples were boiled for 30 min and spined down. 10–15 μl of supernatant was loaded on an SDS-PAGE gel and continued with western blot probed by GFP antibody (Beyotime) (b) and anti-DnaJ antiserum (c) detection. DnaK protein was purified from E. coli BL21 (TaKaRa, China)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3824243&req=5

Fig1: Identification of pAE03-dnaJ-gfp in S. pneumoniae strain D39-dnaJ-gfp. Two positive colonies (D39-dnaJ-gfp) were selected on BA plates supplemented with 0.25 μg/ml erythromycin, and confirmed by PCR with gfp primers (a). The samples were D39 (1), plasmid pAE03 (2), D39-dnaJ-gfp 2 (3), D39-dnaJ-gfp 1 (4) and marker (M).Then they were cultured in C+Y medium until OD600 0.4–0.5. Bacteria was collected by centrifugation at 12,000 rpm and washed twice with PBS. The pellet was then resuspended in 50–80 μl 2× SDS sample buffer. Samples were boiled for 30 min and spined down. 10–15 μl of supernatant was loaded on an SDS-PAGE gel and continued with western blot probed by GFP antibody (Beyotime) (b) and anti-DnaJ antiserum (c) detection. DnaK protein was purified from E. coli BL21 (TaKaRa, China)

Mentions: PCR and sequencing were used to confirm the successful construction of pAE03-dnaJ-gfp (Fig. 1a). The resulting plasmid was transformed into pneumococcal D39 strain, GFP or DnaJ-GFP fusion protein was detected with western blot probed with a 1:300–500 dilution of GFP antibody (Fig. 1b) or a 1:8,000 dilution of anti-DnaJ antiserum (Fig. 1c). When detected with GFP antibody, there was one band with a molecular weight (Mw) ~67 kDa in S. pneumoniae strain D39-dnaJ-gfp; no band could be detected in WT D39 strain (Fig. 1b). The ~67 kDa protein is coincident with the Mw of DnaJ-GFP fusion protein, because DnaJ has the Mw of approximate 40 kDa and the Mw of GFP is approximate 27 kDa. We observed two bands in D39-dnaJ-gfp, but only one band in D39, no band in negative control pure protein DnaK (Fig. 1c) probed with anti-DnaJ antiserum. The lower band indicated DnaJ, and the higher represented fusion protein DnaJ-GFP. Together, these results showed that plasmid pAE03-dnaJ-gfp has been successfully constructed and transformed into D39 which can be used for further studies.Fig. 1


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)

Identification of pAE03-dnaJ-gfp in S. pneumoniae strain D39-dnaJ-gfp. Two positive colonies (D39-dnaJ-gfp) were selected on BA plates supplemented with 0.25 μg/ml erythromycin, and confirmed by PCR with gfp primers (a). The samples were D39 (1), plasmid pAE03 (2), D39-dnaJ-gfp 2 (3), D39-dnaJ-gfp 1 (4) and marker (M).Then they were cultured in C+Y medium until OD600 0.4–0.5. Bacteria was collected by centrifugation at 12,000 rpm and washed twice with PBS. The pellet was then resuspended in 50–80 μl 2× SDS sample buffer. Samples were boiled for 30 min and spined down. 10–15 μl of supernatant was loaded on an SDS-PAGE gel and continued with western blot probed by GFP antibody (Beyotime) (b) and anti-DnaJ antiserum (c) detection. DnaK protein was purified from E. coli BL21 (TaKaRa, China)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Identification of pAE03-dnaJ-gfp in S. pneumoniae strain D39-dnaJ-gfp. Two positive colonies (D39-dnaJ-gfp) were selected on BA plates supplemented with 0.25 μg/ml erythromycin, and confirmed by PCR with gfp primers (a). The samples were D39 (1), plasmid pAE03 (2), D39-dnaJ-gfp 2 (3), D39-dnaJ-gfp 1 (4) and marker (M).Then they were cultured in C+Y medium until OD600 0.4–0.5. Bacteria was collected by centrifugation at 12,000 rpm and washed twice with PBS. The pellet was then resuspended in 50–80 μl 2× SDS sample buffer. Samples were boiled for 30 min and spined down. 10–15 μl of supernatant was loaded on an SDS-PAGE gel and continued with western blot probed by GFP antibody (Beyotime) (b) and anti-DnaJ antiserum (c) detection. DnaK protein was purified from E. coli BL21 (TaKaRa, China)
Mentions: PCR and sequencing were used to confirm the successful construction of pAE03-dnaJ-gfp (Fig. 1a). The resulting plasmid was transformed into pneumococcal D39 strain, GFP or DnaJ-GFP fusion protein was detected with western blot probed with a 1:300–500 dilution of GFP antibody (Fig. 1b) or a 1:8,000 dilution of anti-DnaJ antiserum (Fig. 1c). When detected with GFP antibody, there was one band with a molecular weight (Mw) ~67 kDa in S. pneumoniae strain D39-dnaJ-gfp; no band could be detected in WT D39 strain (Fig. 1b). The ~67 kDa protein is coincident with the Mw of DnaJ-GFP fusion protein, because DnaJ has the Mw of approximate 40 kDa and the Mw of GFP is approximate 27 kDa. We observed two bands in D39-dnaJ-gfp, but only one band in D39, no band in negative control pure protein DnaK (Fig. 1c) probed with anti-DnaJ antiserum. The lower band indicated DnaJ, and the higher represented fusion protein DnaJ-GFP. Together, these results showed that plasmid pAE03-dnaJ-gfp has been successfully constructed and transformed into D39 which can be used for further studies.Fig. 1

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