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Purification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of  Galleria mellonella  silk proteinase inhibitor 2 ( Gm SPI-2) determined both by X-ray diffraction and NMR spectroscopy methods.  Gm SPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of  Gm SPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with  R  factor of 10.57% and  R   free  of 12.91%. The 3D structure of  Gm SPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β 2 αβ fold characteristic for Kazal-family serine proteinase inhibitors.

Atomic Resolution Structure of a Protein Prepared by Non-Enzymatic His-Tag Removal. Crystallographic and NMR Study of GmSPI-2 Inhibitor