TY - JOUR
T1 - Expression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis ribosome recycling factor
AU - Guo, Peng
AU - Zhang, Liqiang
AU - Qi, Zhen
AU - Chen, Runsheng
AU - Jing, Guozhong
N1 - Funding Information:
We thank Professor A. Kaji, Department of Microbiology, School of Medicine, University of Pennsylvania, for providing E. coli LJ14 strain. This work was supported by grants (Nos. G1999075608 and 30170210) from the China Committee for Science and Technology.
PY - 2005/7
Y1 - 2005/7
N2 - A very promising approach to understanding the mechanism of protein thermostability is to investigate the structure-function relationship of homologous proteins with different thermostabilities. Ribosome recycling factor (RRF), which is an essential factor for protein synthesis in bacteria, may be a good candidate for such study. In this report, a ribosome recycling factor from Thermoanaerobacter tengcongensis was expressed and characterized. This protein contains 184 residues, shows 51.4% identity to that of Escherichia coli RRF, and has very strong antigenic cross-reactivity with antibody to E. coli RRF. In vivo activity assay shows that weak residual activity may remain in TteRRF in E. coli cells. Circular dichroism spectral analysis shows that TteRRF has a very similar secondary structure to that of E. coli RRF, implying that they have similar tertiary structures. However, their thermostabilities are significantly different. To find which domain of RRF is mainly responsible for maintaining stability, TteDI/EcoDII and EcoDI/TteDII RRF chimeras were created. Their domain I and domain II are from E. coli and T. tengcongensis RRFs, respectively. The results of GdnHCl and heat induced denaturation of the chimeric RRFs suggest that the domain I plays a major role in maintaining the stability of the RRF molecule.
AB - A very promising approach to understanding the mechanism of protein thermostability is to investigate the structure-function relationship of homologous proteins with different thermostabilities. Ribosome recycling factor (RRF), which is an essential factor for protein synthesis in bacteria, may be a good candidate for such study. In this report, a ribosome recycling factor from Thermoanaerobacter tengcongensis was expressed and characterized. This protein contains 184 residues, shows 51.4% identity to that of Escherichia coli RRF, and has very strong antigenic cross-reactivity with antibody to E. coli RRF. In vivo activity assay shows that weak residual activity may remain in TteRRF in E. coli cells. Circular dichroism spectral analysis shows that TteRRF has a very similar secondary structure to that of E. coli RRF, implying that they have similar tertiary structures. However, their thermostabilities are significantly different. To find which domain of RRF is mainly responsible for maintaining stability, TteDI/EcoDII and EcoDI/TteDII RRF chimeras were created. Their domain I and domain II are from E. coli and T. tengcongensis RRFs, respectively. The results of GdnHCl and heat induced denaturation of the chimeric RRFs suggest that the domain I plays a major role in maintaining the stability of the RRF molecule.
KW - Expression and characterization
KW - Ribosome recycling factor
KW - Thermoanaerobacter tengcongensis
KW - Thermostability
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U2 - 10.1093/jb/mvi102
DO - 10.1093/jb/mvi102
M3 - Article
C2 - 16046452
AN - SCOPUS:23344432648
SN - 0021-924X
VL - 138
SP - 89
EP - 94
JO - Journal of Biochemistry
JF - Journal of Biochemistry
IS - 1
ER -