Cloning, expression, purification and crystallization of Pf-1806301

by 1978- Huang, Lei

Pf -1806301, a putative archaeal Lsm protein characterized in this thesis, shows sequence similarity to both archaeal Lsm and bacteria Hfq proteins. Sm/Lsm proteins are a large family of RNA-binding proteins that have been identified in archaea, bacteria and eukarya. The determination of the three-dimensional structure of Pf -1806301 therefore will not only help reveal the functions of the Lsm proteins, but also offer in-depth implications to the relationship between Lsm and Hfq proteins. In order to solve the crystal structure of Pf -1806301, the gene encoding Pf -1806301 was recloned into vector pET28, which provides a thrombin cleavage site for the removal of the Histag after protein purification. The best Se-Met crystal diffracted up to 2.7Å, showing a significant improvement in diffraction compared with crystals grown from the protein with the His-tag attached. In addition, diffraction data reveal that the Pf -1806301 complex is of a dimer of octamers.