Analysis and Control of Protein Crystallization Using Short Peptide Tags That Change Solubility without Affecting Structure, Thermal Stability, and Function

Short peptide tags attached to recombinant proteins are emerging as an important tool for biochemical, research. Here, we report the effects of 10 Solubilization Controlling Peptide (SCP) tags on crystallization behavior of a bovine pancreatic trypsin inhibitor (BPTI) variant: The tags did not affect structure, thermodynamics, and activities of BPTI. Moreover, eight of the tagged variants crystallized under the same condition, and six of them diffracted at high resolution. All variants with long-term solubility (LS) between. 1 and 6 mg/mL produced crystals that diffracted well, while variants with LS < 1 and >6 mg/mL did not crystallize, produced poorly diffracting crystals, or crystallized under a different condition. The only exception was a glutamine tagged variant, which had an LS of 5 mg/mL, but fast aggregation kinetics, and produced mere needles unsuitable for further analysis. Crystal Structures indicated that most tags were largely invisible, indicating high flexibility, without having interactions with nearby residues. Therefore, short peptides, introducing a mere 5-7 residue elongation, could provide a useful technology for tuning protein solubility without affecting its other properties and hence for overcoming problems associated with excessively low or high solubility, such as in crystallization.