Cloning, Expression, Purification And Preliminary Crystallographic Analysis Of Mj. Nifh1 And Sc. Ndl1

1 Cloning, expression, purification, crystallization and preliminary crystallographic analysis of NifH1 from Methanocaldococcus jannaschiiNitrogen fixation is catalyzed by nitrogenase complex in Azotobacter, which is composed by dinitrogenase and dinitrogenase reductase. Dinitrogenase is anα2β2 heterotetramer of the proteins NifD and NifK. Dinitrogenase reductase is a homodimer of the protein NifH. The expression of NifD/K and NifH nitrogenase homologs (named NflD/K and NflH for'Nif-like'D and H, respectively) have been detected in a non-nitrogen-fixing hyperthermophilic methanogen Methanocaldococcus jannaschii, but the function is unknown. Solving the structure of Mj.NifH1 may help us to better understand its function and supply some clues to understand the evolution of nitrogenase.In this work, we constructed the expression vectors containing the cDNA sequence of NifH1 from M. jannaschii, and expressed the full length protein with an additional His6 tag at the C-terminus. Then the target protein was further purified by Nickel-chelating column and HiLoad 16/60 Superdex 200 size-exclusion column and crystallized by the hanging-drop vapour-diffusion method at 287K. An X-ray diffraction data set was collected at a resolution of 3.3 ?. The crystal belonged to space group P4132, with unit-cell parameters a = b = c =139.45 ?, and was estimated to contain one protein molecule per asymmetric unit.2 Cloning, expression, purification and crystallization of Ndl1 from Saccharomyces cerevisiaeDynein is a minus-end-directed microtubule motor, which can transform chemical energy include ATP into mechanical energy in cell. The critical roles of dynein in mitosis, membrane transport and intracellular transport need the cooperation of some protein. Ndl1, one of the family NUDEL,is required in the process of location of dynein into kinetochore. Ndl1 can activate the transportation of dynein-mediated protein and stabilize kinetochore dynein/dynactin to facilitate the force generation function of the motor. The loss of Ndl1 will reduce the dynein associating with the plus end of microtubules, which leads to inefficient mitosis.In this work, we constructed the expression vectors containing the cDNA sequence of Ndl1 from Saccharomyces cerevisiae, and the full length protein was expressed and purified. We got a protein crystal of Sc.Ndl1 though the method of hanging-drop vapour-diffusion at 287K. The optimization of crystal is in process.