Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance.
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 analyze the possible function of this LMM HSP under heat stress. 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 the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl beta-D-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5 degrees C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55 degrees C, 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 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5 degrees C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.
Yeh
C H
CH
Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China.
Chang
P F
PF
Yeh
K W
KW
Lin
W C
WC
Chen
Y M
YM
Lin
C Y
CY
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics*; D018869:Heat-Shock Response / Q000235:genetics*; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics
D006360:Heat-Shock Proteins; C108681:Oshsp16.9 protein, Oryza sativa; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase