Analysis and Control of Protein Crystallization Using
Short Peptide Tags That Change Solubility without Affecting Structure,
Thermal Stability, and Function
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Abstract
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 (<i>LS</i>) between
1 and 6 mg/mL produced crystals that diffracted well, while variants
with <i>LS</i> < 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 <i>LS</i> 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