Expression, Purification And Crystalization Of Pin1 Protein

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivitol signaling mechanism in diverse cellular processes and its deregulation can lead to human disease. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. Peptidyl-prolyl isomerase Pin1 is an essential protein in regulating cell entry into mitosis by catalyzing the conformational change of many critical proteins. So far, more than 20 Pin1 targets have been isolated, such as cdc25, Weel, Mytl, cdc27, cyclin D1 andβ-catenin, suggesting that Pin1 might play an important role in the cell cycle regulation,signal transduction and human disease. Overexpression of Pin1 is prevalently found in human cancers, whereas its inhibition induces apoptosis and contributes to the neuronal death in Alzheimer's disease.Pin1 is a protein of 18KD, with two-domain structure, consisting of an N-ter- mianal WW domain and a C-terminal PPIase domain, which together form a double -check mechanism. The WW domain binds to only specific pSer/Thr-Pro motifs that are oftencrucial regulatory phosphorylation sites in Pin1 substrates. The binding of WW domain targets Pin1 to its substrates, whereas its PPIase domain isomerizes spe- cific pSer/Thr-Pro motifs to induce conformational changes. Although the structure of Pin1 has first been determined in 1997, and studies on its structure basis has also been on focusing on the interaction of Pin1 with its short peptide substrate (4 to 7 amino acid residues), very little is known about how Pin catalyzes its reaction, and how Pin1 interacts with its biological substrates. Therefore, in the current studies, We desinged wo mutants in each domain (W34A&K63A) and expressed in E.coli BL21(DE3). The two-step purification by affinity chromatography and FPLC (fast performance liquid chromatography) were developed to yield wild and mutant Pin1 protein. Crystals of wild and mutant Pin1, Pin1/cdc25 complex were obtained by the hanging drop vapor diffusion method and many different crystals forms were observed.The result indicate that crystal of W34A belongs to space group P4(3)2(1)2,with unit-cell parameters P3(1)21 a=b=49.011 (A|°);c=136.796 (A|°),α=β=γ=90 and diffracted to 2.5 (A|°). crystal of K63A belongs to space group P3(1)21 ,with unit-cell parameters a=b=68.646 (A|°);c=79.412 (A|°),α=β=90,γ=120,and diffracted to 2.6 (A|°).Meanwhile The conformation of protein Pin1-wildtype ,pin1-W34A and Pin1-K63A were analyzed by circular dichroism (CD) analysis.we found there were no significant differences between them. We hope it would provide a structural basis for rational drug design.