Expression, Purification And Crystallization Of Plant Chloroplast Division Protein ARC6 And PDV2

Chloroplast is a type of organelles wrapped by a bilayer membrane in plant cells, and the physiological functions of chloroplast include regulating plant growth and reproduction, etc. Chloroplast proliferates via a division manner, in which process many protein complexes play important functions. ARC6 is a single-pass transmembrane protein located at the division site of chloroplast, with its N-terminal towards the stroma side. Specifically, the short C-terminal of ARC6 interacts with PDV2 in the intermembrane space. The interaction of ARC6-PDV2 connects the chloroplast division machineray from inside to the outside of the chloroplast. However, the underlying mechanism of this remains unknown, and is the current research focus.In this study, we successfully cloned three genes ARC6 (646-801aa), PDV2 (236-307aa) and ARC6 (76-618aa), and they were properly expressed and purified in E.coli through His-tag and GST-tag purification methods.We obtained the protein complexes between ARC6 (646-801aa) and PDV2 (236-307aa). Subsequently, we got six conditions under which crystals can grow. Through continuous optimization experiments, we obtained high resolution diffraction data using the crystals under the HR2-112-3# condition. Moreover, we got the crystal of selenomethionine-substituted ARC6 (646-801aa) protein, and the diffraction data and the crystal phase information were obtained finally. The crystal structure of ARC6 (646-801aa) has been resolved. In addition, we have conducted researches to obtain the structure of the protein complexes of ARC6 (674-801aa) and PDV2 (280-307aa) by co-crystallization methods. Currently one condition under which crystals can grow is found.On the other side, we have got the optimized ARC6 (76-618aa) crystal with two different conditions after purification and crystallizations. The diffraction studies of the crystal is undergoing presently.