Optimal 1TEL-target protein linker character is target protein-dependent

Fusing a variant of the sterile alpha motif domain of the human translocation ETS leukaemia protein (1TEL) to a protein of interest has been shown to significantly enhance its crystallization propensity. 1TEL is a pH-dependent, polymer-forming protein crystallization chaperone which, when covalently fused to a protein of interest, forms a stable, well ordered crystal lattice. However, despite its success, a challenge persists in that crystal quality and diffraction limits appear to be heavily dependent on the choice of linker between 1TEL and the protein of interest, with the identification of a functional linker currently relying on trial-and-error methods. Likewise, previous studies revealed that a ten-histidine tag at the 1TEL N-terminus can either facilitate or hinder the ordered crystallization of target proteins attached via flexible or semi-flexible linkers. To address these challenges, we designed multiple constructs with several types of linkers [rigid (helical fusion), semi-flexible (Pro-Ala and Pro-Ala-Ala) and flexible (Gly-Gly and Gly-Gly-Gly)] of varying lengths to fuse either a designed ankyrin-repeat protein (DARPin) or the thirty-eight-negative kinase-1 ubiquitin-associated (UBA) domain to the 1TEL C-terminus. Semi-flexible and flexible linker constructs were made with and without a ten-histidine tag. Our findings indicate that short semi-flexible and rigid linkers consistently yielded large crystals with a DARPin target protein, but that flexible linkers performed best with a UBA-domain target protein. Removing the ten-histidine tag uniformly enhanced crystallization rates, improved the crystal morphology and increased the crystallization propensity of the semi-flexible and flexible linker constructs. These results suggest that the ideal linker selection primarily depends on the properties of the target protein. Our data support our current recommendation to use a short flexible or semi-flexible linker between 1TEL and the target protein to facilitate protein crystallization and high-resolution structure determination.