Prokaryotic Expression, Purification And Thermal Stability Of HPin1 And Its Interaction With Al～(3+)

Pinl is a kind of peptidyl-prolyl cis/trans isomerase. Pinl specifically isomerizes the pSer/Thr-Pro motifs in proteins, relating to kinds of human diseases, especially cancer and AD. The His-tagged hPinl was expressed by the pET-19b-hPinl plasmid in E.coli BL21 and was purified using the chelating sepharose column. The N-terminal fusion His-tag was cleaved offby the recombinant enterokinase. Thermal stability of hPinl, and its interaction with Al³⁺ had been intensively studied for the first time, by means of SDS-PAGE, UV difference spectra, intrinsic fluorescence spectra, circular dichroism spectra and PPIase activity assays.The thermal stability of His-hPinl was investigated. Heat treatment induced some of His-hPinl dissolvability at 50℃～100℃. The maximum fluorescence emission of the His-hPinl or hPin1 was at 340nm, suggesting that tryptophan residues were located in a hydrophobic environment of the hPin1 molecule. Heat treatment resulted in shift of the maximum emission wavelength and decrease of the fluorescence emission intensity, suggesting some conformation changes of hPin1. Further, the far-UV circular dichroism results showed that secondary structure of hPin1 was altered after heat treatment. The content of a-helix remarkably decreased with increase of random coil. The PPIase activity of hPin1 remained the same when temperature was below 40℃, but decreased when temperature was higher than 50℃. All the results suggested that hPinl is a relative thermal stability protein.A gradual increase in absorbance values of LMCT bands of the Al³⁺-hPin1 system was observed with time-scanning UV absorption spectra. The IF intensity of hPin1 was quenched by Al³⁺ in a static manner; the Stern-Volmer quenching constant K_SV is 9.14×10³ (mol/L)^-1·s^-1, and a pronounced "red" shift inλ_em,max for the IF emission spectra (excited at 295 nm) was detected, which indicated the degree of solvent exposure of the tryptophan residues in hPinl increased in the presence of Al³⁺. The CD spectra provided evidence that the binding of Al³⁺ induced hPin1 to undergo a rearrangement of the secondary structural elements, exhibiting a decrease in a-helix and increases inβ-sheet and unordered coil. What's more, the PPIase activity of hPinl decreased in the presence of Al³⁺. The results showed that hPin1 can conjugate with Al³⁺ in vitro, resulting in that the structure of hPin1 encountered some changes, and the PPIase activities of hPin1 reduced significantly. These results suggest that the deregulation of hPin1 induced by Al³⁺ may be a putative reason for the formation of tangles and plaques in the pathogenesis of AD.