Purification and characterization of Cyclin-H1 from Arabidopsis thaliana

Cyclin H (CycH), a member of the large cyclin family, participates in every process of cell division. Its biological functions and importance have received wide attention in mammalians, but not in higher plants. This work reports a protein purification protocol for obtaining Arabidopsis CycH;1 (AtCycH;1) from prokaryotic expression system, followed by characterization of its biophysical properties. The protein was constructed with a His-tag at its N-terminus. One-step nickel-affinity purification yielded high pure target protein, which behaved as a monomer in the testing condition. Circular Dichroism spectrum revealed that AtCycH;1 is a helical protein containing a significant amount of disordered structures. Further assays indicated that AtCycH;1 exhibits poor heat-resistance and can be easily degraded in room temperature, suggesting low stability for the protein. The flexible and unstable properties may be intrinsic to the protein in vivo as it has to bind with different partners during the cell cycle and be promptly degraded to meet the phase transition. The instability, however, can be improved by adding SO4(2-) ion in the protein buffer. The presence of a high concentration of SO4(2-) is capable of increasing the thermal stability and inhibiting the degradation. Irrespective of whether the association of SO4(2-) with AtCycH;1 drives the protein into more compact form or not, the current results may provide clues for a successful crystallization of AtCycH;1 and its subsequent structural analysis in the future.