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Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein

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Abstract

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His6-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro.

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Abbreviations

CS:

citrate synthase

ER-sHSP:

endoplasmic reticulum-located small heat shock protein

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IPTG:

isopropyl-1-thio-β-d-galacto-pyranoside

LDH:

lactate dehydrogenase

ME:

mercaptoethanol

PVPP:

Polyvinylpolypyrrolidone

sHSP(s):

small heat shock protein(s)

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Acknowledgments

This work was supported in part by funds from the Bio-oriented Technology Research Advancement Institution. We thank Jennifer Calcaterra and Drs. Kempton Horken and Hasanova Gulnara at the University of Nebraska-Lincoln (USA) for a critical reading of the manuscript.

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Authors and Affiliations

  1. Tropical Agriculture Research Front, Japan International Research Center for Agricultural Sciences (JIRCAS), 1091-1 Maezato Kawarabaru, Ishigaki, Okinawa, 907-0002, Japan

    Tarlan G. Mamedov & Mariko Shono

  2. Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Othmer Hall 820 N 16 St, Lincoln, NE, 68588-0668, USA

    Tarlan G. Mamedov

Authors
  1. Tarlan G. Mamedov
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  2. Mariko Shono
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Correspondence to Tarlan G. Mamedov.

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10265_2008_148_MOESM1_ESM.doc

Supplementary Fig. 1. Immunoblot analysis of tomato plant expressing LeHSP21.5 protein. Tomato plant growing and subcellular fractionation was performed as described in Materials and Methods. Ten microliters of total proteins from each fraction were run on 12% SDS-PAGE and transferred to PVDF membrane. Immunoblotting was performed using anti-LeHSP21.5 polyclonal antibody. 1: 25°C, 10,000x g supernatant; 2: 25°C, 200,000× g pellet; 3: 25°C, 200,000× g supernatant; 4: 40°C, 10,000× g supernatant; 5: 40°C, 200,000× g pellet; 6: 40°C, 200,000× g supernatant. (DOC 378 kb)

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Mamedov, T.G., Shono, M. Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein. J Plant Res 121, 235–243 (2008). https://doi.org/10.1007/s10265-008-0148-x

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  • DOI: https://doi.org/10.1007/s10265-008-0148-x

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