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Functional characterization of a small heat shock protein from Mycobacterium leprae.

Lini N, Rehna EA, Shiburaj S, Maheshwari JJ, Shankernarayan NP, Dharmalingam K - BMC Microbiol. (2008)

Bottom Line: Physical interaction of the chaperone with target protein is also demonstrated.The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins.Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Tamil Nadu, India. lininirmala@gmail.com

ABSTRACT

Background: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty.

Results: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins.

Conclusion: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

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(A). sHsp18 interacts with SmaI. Heat inactivated SmaI was added to, sHsp18 bound MagneHis particles as per methods. sHsp18 was able to interact and refold the heat inactivated SmaI and restore its biological activity. But in the control, heat inactivated SmaI without sHsp18 did not show any activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid DNA digested with heat inactivated SmaI added to sHsp18 bound MagneHis particles (lane 3), plasmid DNA treated with heat inactivated SmaI added to MagneHis particles (lane 4). (B) SmaI do not bind directly to MagneHis particles. Native SmaI was incubated with MagneHis particles for 5 min and the supernatant and pellet fractions were taken for restriction assay. Lanes represent, λ Hind III marker (lane 1). Plasmid DNA was digested with SmaI added MagneHis pellet fraction (lane 2), plasmid DNA digested with supernatant fraction (lane 3) and control digest (lane 4).
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Figure 4: (A). sHsp18 interacts with SmaI. Heat inactivated SmaI was added to, sHsp18 bound MagneHis particles as per methods. sHsp18 was able to interact and refold the heat inactivated SmaI and restore its biological activity. But in the control, heat inactivated SmaI without sHsp18 did not show any activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid DNA digested with heat inactivated SmaI added to sHsp18 bound MagneHis particles (lane 3), plasmid DNA treated with heat inactivated SmaI added to MagneHis particles (lane 4). (B) SmaI do not bind directly to MagneHis particles. Native SmaI was incubated with MagneHis particles for 5 min and the supernatant and pellet fractions were taken for restriction assay. Lanes represent, λ Hind III marker (lane 1). Plasmid DNA was digested with SmaI added MagneHis pellet fraction (lane 2), plasmid DNA digested with supernatant fraction (lane 3) and control digest (lane 4).

Mentions: Histidine tagged sHsp18 was bound to MagneHis particles as described in experimental procedures. One dimensional gel analysis confirmed the presence of only sHsp18 protein bound with MagneHis particles. After removal of non specific proteins, heat inactivated SmaI was added to the sHsp18 bound MagneHis particles and incubated at 37°C for 30 min. The mix was washed extensively to remove nonspecifically bound SmaI. After washing, substrate plasmid DNA was added to the magnetic beads containing bound sHsp18-SmaI complex. As shown in Fig. 4A, the plasmid DNA was cleaved, indicating that SmaI was retained on the beads presumably through its interaction with sHsp and also regained its biological activity due to the chaperone function of sHsp18. In the control experiment, incubation of heat denatured SmaI with MagneHis particles, without bound sHsp18, did not result in binding of the restriction enzyme. Similarly, native SmaI under the same experimental conditions failed to bind to the MagneHis particles. These control experiments rule out the possibility of nonspecific binding of SmaI to MagneHis particles (Fig. 4B). However, SmaI, heat inactivated at and above 45°C, was unable to bind to sHsp18 (data not shown), which implies that completely denatured proteins could not be refolded to active enzyme by sHsp18.


Functional characterization of a small heat shock protein from Mycobacterium leprae.

Lini N, Rehna EA, Shiburaj S, Maheshwari JJ, Shankernarayan NP, Dharmalingam K - BMC Microbiol. (2008)

(A). sHsp18 interacts with SmaI. Heat inactivated SmaI was added to, sHsp18 bound MagneHis particles as per methods. sHsp18 was able to interact and refold the heat inactivated SmaI and restore its biological activity. But in the control, heat inactivated SmaI without sHsp18 did not show any activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid DNA digested with heat inactivated SmaI added to sHsp18 bound MagneHis particles (lane 3), plasmid DNA treated with heat inactivated SmaI added to MagneHis particles (lane 4). (B) SmaI do not bind directly to MagneHis particles. Native SmaI was incubated with MagneHis particles for 5 min and the supernatant and pellet fractions were taken for restriction assay. Lanes represent, λ Hind III marker (lane 1). Plasmid DNA was digested with SmaI added MagneHis pellet fraction (lane 2), plasmid DNA digested with supernatant fraction (lane 3) and control digest (lane 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629775&req=5

Figure 4: (A). sHsp18 interacts with SmaI. Heat inactivated SmaI was added to, sHsp18 bound MagneHis particles as per methods. sHsp18 was able to interact and refold the heat inactivated SmaI and restore its biological activity. But in the control, heat inactivated SmaI without sHsp18 did not show any activity. Lanes represent, λ Hind III marker (lane 1), undigested plasmid (lane 2), plasmid DNA digested with heat inactivated SmaI added to sHsp18 bound MagneHis particles (lane 3), plasmid DNA treated with heat inactivated SmaI added to MagneHis particles (lane 4). (B) SmaI do not bind directly to MagneHis particles. Native SmaI was incubated with MagneHis particles for 5 min and the supernatant and pellet fractions were taken for restriction assay. Lanes represent, λ Hind III marker (lane 1). Plasmid DNA was digested with SmaI added MagneHis pellet fraction (lane 2), plasmid DNA digested with supernatant fraction (lane 3) and control digest (lane 4).
Mentions: Histidine tagged sHsp18 was bound to MagneHis particles as described in experimental procedures. One dimensional gel analysis confirmed the presence of only sHsp18 protein bound with MagneHis particles. After removal of non specific proteins, heat inactivated SmaI was added to the sHsp18 bound MagneHis particles and incubated at 37°C for 30 min. The mix was washed extensively to remove nonspecifically bound SmaI. After washing, substrate plasmid DNA was added to the magnetic beads containing bound sHsp18-SmaI complex. As shown in Fig. 4A, the plasmid DNA was cleaved, indicating that SmaI was retained on the beads presumably through its interaction with sHsp and also regained its biological activity due to the chaperone function of sHsp18. In the control experiment, incubation of heat denatured SmaI with MagneHis particles, without bound sHsp18, did not result in binding of the restriction enzyme. Similarly, native SmaI under the same experimental conditions failed to bind to the MagneHis particles. These control experiments rule out the possibility of nonspecific binding of SmaI to MagneHis particles (Fig. 4B). However, SmaI, heat inactivated at and above 45°C, was unable to bind to sHsp18 (data not shown), which implies that completely denatured proteins could not be refolded to active enzyme by sHsp18.

Bottom Line: Physical interaction of the chaperone with target protein is also demonstrated.The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins.Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Tamil Nadu, India. lininirmala@gmail.com

ABSTRACT

Background: Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty.

Results: The gene encoding Mycobacterium leprae small heat shock protein (sHsp18) was amplified from biopsy material of leprosy patients, and cloned and expressed in E. coli. The localization and in vitro characterization of the protein are detailed in this report. Data show that major portion of the protein is localized in the outer membrane of E. coli. The purified sHsp18 functions as an efficient chaperone as shown by their ability to prevent thermal inactivation of restriction enzymes SmaI and NdeI. Physical interaction of the chaperone with target protein is also demonstrated. Size exclusion chromatography of purified protein shows that the protein can form multimeric complexes under in vitro conditions as is demonstrated for several small heat shock proteins.

Conclusion: The small heat shock protein sHsp18 of M. leprae is a chaperone and shows several properties associated with other small heat shock proteins. Membrane association and in vitro chaperone function of sHsp18 shows that the protein may play a role in the virulence and survival of M. leprae in infected host.

Show MeSH
Related in: MedlinePlus