Expression Of CDK2in Pichia Pastoris And Preliminary Crystallographic Studies

Protein kinases are a group of proteins playing an important role in signaltransduction pathways. They are activated by external signals and pass the information todownstream targets through phosphorylation. Like other kinases, CDK2is activated afterbinding specific cyclins and then takes part in the cell cycle regulation. It has been shownthat over-expression of CDK2is involved in tumorigenesis.In this work, we used two expression systems, Escherichia coli (E. coli) and Pichiapastoris (P. pastoris) to express CDK2. After expression, the target protein was purifiedand analyzed by qualitative and quantitative methods. The P. pastoris system was chosenfor large expression due to better expression level and higher protein stability. The proteinover-expressed in P.pastoris was used to grow crystals. After several trials, the apo CDK2crystals were obtained.The results and procedures are listed as following:1. The cdk2cDNA was amplified by PCR and used to construct the prokaryoticexpression vector pET28a(+)-GST-TEV-cdk2and the eukaryotic expression vector(pPIC3.5K-cdk2, pPIC9K-cdk2). The plasmid pET28a(+)-GST-TEV-cdk2wastransformed into BL21(DE3) Rosetta for intracellular expression. The plasmidspPIC3.5K-cdk2and pPIC9K-cdk2were used for intracellular and extracellularexpression in P. pastoris respectively. Then the two plasmids were linearized andtransformed into P. pastoris GS115by electrotransformation. Then the target proteinexpression was induced with methanol.2. The target protein was purified using Ni affinity and gel filtration chromatography.The resulting fractions were resolved by SDS-PAGE and the identity of the proteinwas confirmed by Western-Blot. The soluble form of CDK2was expressed in bothsystems, with the yields of4.68mg/L and6.04mg/L in E. coli and P. pastorisrespectively. However, no product was detected for extracellular expression. To exam the protein folding, the proteins from both systems were analyzed by CircularDichroism. The results indicated that the percentage of correctly fold CDK2was63.9%in P. pastoris and13.7%in E. coli.3. The protein expressed in P. pastoris was purified and the crystallization trials were setup with commercial screening kits. The crystals of apo CDK2were obtained after twodays’ incubation at4degree.The results of this study showed that CDK2can be expressed in P. pastoris efficiently.The P. pastoris eukaryotic expression system provides a good platform for the large-scaleexpression of functionally important proteins. It laid good foundation for the subsequentcrystallization and inhibitor screening efforts.