Expression,purification And Preliminary Study Of Structural Biology Of The Truncated AtTic110 At The Chloroplast Inner Envelope Membrane Of Arabidopsis

Chloroplast translocon is the important component to mediate the entry of precursor proteins into chloroplast.The translocon at the outer and inner envelope of chloroplast are respectively termed TOC and TIC.Tic110,a component of TIC complex,had been proposed to be transmembrane channel-like protein across the inner envelope membrane;however,the evidence is still insufficient.So far,the three-dimensional structure of Tic110 is not clear,and its position in the inner membrane is ambiguous.Thence,in this study,we constructed multiple protein truncations of the long C-terminal region of Arabidopsis thaliana Tic110(AtTic110).After heterologous expression in E.coli BL21(DE3),nickel ion affinity chromatography,heparin affinity chromatography,and gel filtration chromatography were used to purify different truncated proteins,and the protein which with better properties was preliminarily screened for crystallization conditions and observed under negative staining electron microscopy.The preliminary results obtained are as follows:(1)Eight AtTic110?N recombinant expression vectors were successfully constructed and expressed in E.coli.Two truncated proteins with better properties were obtained,namely AtTic110(143-700)and AtTic110(143-1016).(2)0.6?1.0 mg AtTic110(143-700)protein sample can be obtained per liter of bacterial liquid.Through preliminary screening of AtTic110(143-700)protein crystallization conditions,four crystallization conditions with relatively good crystal shapes were obtained.However,due to the small size of the crystal,X-ray diffraction has not yet been performed,and further optimization is needed in the later stage.(3)1.0?1.5 mg AtTic110(143-1016)protein sample can be obtained per liter of bacterial liquid.After preliminary screening of crystals,protein crystals have not yet been obtained.However,the negative staining results of transmission electron microscopy show that the properties of the protein are better,and can try to use cryo-electron microscopy for follow-up research.