Expression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis ribosome recycling factor.
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.
Guo
Peng
P
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, PR China.
Zhang
Liqiang
L
Qi
Zhen
Z
Chen
Runsheng
R
Jing
Guozhong
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005816:Genetic Complementation Test; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D046968:Thermoanaerobacter / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C087267:ribosome releasing factor