Optimising His-tags for purification and phasing

Abstract

Several affinity protein tags consisting of short peptides or whole domains have been developed, which are included as an additive protein sequence on the C- or/and N-terminus. Their highly specific strong affinity affords a one-step purification with minimal effect on biological activity. On the other hand, protein tags are often disordered in the crystal structure and complicate protein crystallisation. Experimental phasing of macromolecules by anomalous dispersion requires well ordered atoms in the crystal lattice. A pre-organised protein tag with metal chelating properties can bind anomalous scatterers in a predefined way and enhance SAD and MAD data quality using synchrotron radiation. As a welcome side-effect such protein tags could adopt stable conformation in crystals and help the crystallisation process. Here we report polypeptides synthesised by solid phase peptide synthesis which may prove useful for both metal affinity chromatography and macromolecular phasing as well. Their chelating properties could be proved by metal ion affinity chromatography. A distinct secondary structure is induced by metal ion chelation. The beta-hairpin structure could be confirmed by circular dichroism spectroscopy and NMR. Fusion proteins made of the Maltose-binding protein fused with our polypeptides were used to check IMAC behaviour under real conditions and to validate crystal growth promotion. First crystal structures from native protein could be solved but shows partly unfolded protein tags. To separate the structural impact of the fusion protein on the tag various linker sequences were taken into account.