Studies On The Folding Of The Protein Tyrosine Phosphatase From Thermus Thermophilus HB27

In this research, we have cloned the full-length sequence encoding PTPase gene from the genomic DNA of T. thermophilus HB27 and expressed the active and soluble recombinant PTPase successfully in E. coli. After a simple purification procedure, we obtained this thermostable enzyme, and then characterized its biochemical and unfolding properties in detail in the following study. Our findings are stated below:1. With the aids of bioinformatics, the pI and molecular weight were estimated to be 5.88 and 22.26 kDa, respectively. There were some differences in amino acid frequences between PTPase of T. thermophilus HB27 and Swiss-Port protein sequence database. The content of polar amino acids was 55.8%. The maximum and minimum of hydrophobicity were 2.433 and-2.222, respectively. The amino acid sequences at the positions of 2～18,82-90 and 140-145 were hydrophobic. The sequence alignment indicated that there was 100% similarity in amino acid sequences between PTPase of T. thermophilus HB27 and Tt1001 of T. thermophilus HB8, but had only 30.5% similarity with PTPase (AAB23588) of rats liver. Three types of secondary structuresα-helix,β-sheet and random coil) existed in PTPase according to the secondary structure prediction. Phylogenetic tree analysis showed that PTPase were evolved into two types of branches in the process of evolution:microbial PTPase and PTPase of plants and animals.2. The effects of pH, temperature and ions on the activity of PTPase were studied when using pNPP as the substrate. The results showed a optimum pH range of 3.6～4.0 and optimum temperature of 75℃. k_cat/K_m were 1.77*10⁴ M^-1 and 6.68*10⁵ M^-1·s^-1at 30℃and 75℃, respectively. Its activity could be activated by Mn²⁺, followed by the order of Mg²⁺, Ca²⁺, Ba²⁺and Ni²⁺, but inhibited by Zn²⁺, Cu²⁺, Cl" and SO₄^2-. These results suggested that PTPase might play important physiological roles in vivo to adapt T. thermophilus HB27 to the extreme temperatures and specific nutritional conditions.3. The activity and conformational changes of PTPase were investigated in the presence of urea, GdnHCl and SDS. The results showed the activity of PTPase was inactivated by these denaturants in a concentration and time-dependent manner. The IC₅₀ value were 2.65 M,0.24 M and 11.27μM, respectively. Inactivation kinetics revealed that all of them were reversible mixed-type inhibition and the inactivation was mono-phase process. Urea and GdnHCl denaturation were showed to be a line in the secondary plot, while SDS was a parabola. Spectral analysis indicated that the unfolding of PTPase induced by urea was a two-state transition whereas in the case of GdnHCl, intermediate was induced and stabilized at lower concentrations. At lower concentration, SDS induced the changes of the tertiary structures and the increase of a-helix structures of PTPase. The high content of secondary structure was stabilized by higher concentration SDS.4. The activity of PTPase was significantly inhibited by Cu²⁺and Zn²⁺in a concentration-and time-dependent manner. TheiC₅₀ value were 15μM and 8.34 mM, respectively. Sequential kinetic studies suggested that both of them induced a reversible mixed-type inhibition and the inactivation was a mono-phase process. The spectral studies showed that both of them induced the tertiary conformational changes of PTPase, but did not change its secondary structures. The PTPase inactivated by Cu²⁺could not be reactivated by EDTA, but reactivated up to 40%of native activity with DTT treatment. Probably, the cysteine residues located around the active sites of PTPase were oxidized by Cu²⁺, which further resulted in the inactivation and the tertiary conformational changes of PTPase.In conclusion, this study is useful to understand its heat-resistant mechanism, folding mechanism, structure and function of this new thermostable PTPase, as well as the construct of high efficient and heat-tolerant engineering strains and their possible applications in industry. In addition, it is meaningful to develop the drugs with PTPase as the targets in order to treat human diseases with relation to PTPase with the inhibitors of PTPase.