Molecular analysis of the structure-Function relationship of Rice (Oryza sativa) class I low molecular mass heat shock protein, Oshsp16.9
1999
Hochschulschrift
Zugriff:
87
A gene encoding the rice 16.9 kDa class I low molecular mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to obtain large amount of the fusion protein and analyze the physiological functions of this LMM HSP. We found that the 16.9 kDa HSP collected by cleavage of the recombinant protein could also form a protein complex of ~310 kDa under non-denaturing conditions as can the small, native, class I HSP from heat-shocked rice seedlings. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-blue cells transformed with a recombinant plasmid containing a glutathione s-transferase (GST)-Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with pGEX-2T vector (pGST cells) under heat shock (HS) after isopropyl b-D-thiogalactopyranoside (IPTG) induction. The pGST-FL cells demonstrated thermotolerance at 47.5oC, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55oC, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 amino acids in the Oshsp16.9 portion), but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 amino acids in the Oshsp16.9 portion), pGST-N72 cells (cells produced a fusion protein with the N-terminal 72 amino acids in the Oshsp16.9 portion), pGST-FLD59-78 cells (cells produced a fusion protein without the consensus II region in the Oshsp16.9 portion), or pGST-N78D1-21(cells produced a fusion protein as GST-N78 with the first 21 N-terminal residues removed.). Analysis by non-denaturing gradient acrylamide gel revealed the fusion protein expressed in pGST-FL cells and pGST-N78 cells tended to form a specific 3-monomer protein complex. Taken together, the data suggest the consensus II region, especially the sequence EEGNVL (amino acid residues 73 to 78) and the region of 22 and 42 amino acids are crucial for Oshsp16.9 function. Both of these two regions are indispensable for thermoprotection of Oshsp16.9.
Titel: |
Molecular analysis of the structure-Function relationship of Rice (Oryza sativa) class I low molecular mass heat shock protein, Oshsp16.9
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Autor/in / Beteiligte Person: | Yeh, Ching-Hui ; 葉靖輝 |
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Veröffentlichung: | 1999 |
Medientyp: | Hochschulschrift |
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