The thyroid gland is an endocrine gland that synthesises thyroid hormones, which regulate cellular metabolism, growth, and development. μ-crystallin (CRYM) is a cytosolic protein that functions as an intracellular binding protein for the thyroid hormone triiodothyronine (T3), while also exhibiting enzymatic activity as an NADPH-dependent ketimine reductase. By binding T3, it regulates its local availability within cells and thereby its access to the nucleus where its primary function is the regulation of gene expression. Due to its important physiological role, dysfunction of CRYM has been associated with various pathological conditions.
The aim of this thesis was to produce pure recombinant CRYM protein in a bacterial expression system (E. coli). Following the induction of expression, we optimised purification using Ni-affinity chromatography. The thermal stability of CRYM was then characterised under different pH conditions and in the presence of natural ligands (T3, T4, NADH, NADPH) using nanoDSF and DLS methods.
We found that CRYM was significantly more stable under slightly basic conditions (pH 8,0), where it adopted its biologically active homodimeric form and exhibited a higher melting temperature (T$_m$), whereas in acidic environment (pH 5,0–7,0), aggregation occurred. The results of the ligand-binding analysis on CRYM stability were inconclusive, as the protein formed higher oligomeric states, which prevented the ligands from accessing the binding sites. To obtain definitive results, future analysis should be repeated under conditions that ensure the native homodimeric form of the protein.
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