Expression Purification And Characterization Of CyclinH From Arabidopsis Thaliana

CyclinH, a member of the large Cyclin family, tightly participates in every process of cell division. Its biological functions and importance have been received a wide attention in mammalians but not in higher plants. This work reports a protein purification protocol for obtaining high yield and purity Arabidopsis CyclinH from in prokaryotic expression system, followed by characterization of 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 At Cyclin H is a helical protein containing a significant amount of disordered structures. Further assays indicated that AtCyclinH is a poor heat-resistant protein and can easily degrade in room temperature, suggesting instability of the protein. The flexible and unstable properties may be intrinsic for the protein in vivo as it has to bind with different partners during cell cycle and promptly be degraded in order to meet the phase transition. The instability, however, can be improved by adding SO4 2-, PO43- ion and heavy metal ion(Pt2+) in the protein buffer. The presence of high concentration of SO4 2- and PO43- ion is capable of increasing the thermal stability and inhibiting its degradation. And the Pt2+, SO4 2- ion could protect the At Cyclin H from chymotrypsin enzyme digestion. Disregarding whether association of SO4 2- ion with At Cyclin H drives the protein into more compact form or not, current results may provide clues for a successful AtCyclinH crystallization and the following structural analysis in the future.