Polyol Additives Modulate the In Vitro Stability and Activity of Recombinant Human Phenylalanine Hydroxylase

Abstract

Phenylketonuria (PKU; OMIM 261600), the most common disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches. Enzyme replacement therapy with hPAH could be regarded as a potential form of PKU treatment if the reported in vitro hPAH instability could be overcome. In this study, we investigated the effect of different polyol compounds, e.g. glycerol, mannitol and PEG-6000 on the in vitro stability of purified hPAH produced in a heterologous prokaryotic expression system. The recombinant human enzyme was stored in the presence of the studied stabilizing agents at different temperatures (4 and -20 degrees C) during a 1-month period. Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50% glycerol or 10% mannitol, at -20 degrees C, protected the enzyme from loss of its enzymatic activity. The determined DeltaG(0) and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.

Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One Derivatives

“…Recombinant full-length wild-type (WT) hPAH was expressed in E. coli Top10 cells in fusion with a hexa-histidyl peptide to allow purification by immobilized metal affinity chromatography (IMAC) using a Ni-NTA agarose (Qiagen; Hilden, Germany) essentially as described previously [16]. After IMAC purification, hPAH tetramers were isolated by size-exclusion chromatography (SEC) on a HiLoad Superdex 200 HR column (1.6 × 60 cm, GE Healthcare; Chicago, IL, USA) equilibrated with 20 mM Na-HEPES buffer containing 200 mM NaCl, pH 7.0 (SEC buffer), at a flow rate of 0.7 mL/min, at 4 • C (Supplementary Figure S1, peak 3) [13].…”

Protection Against Protein Aggregation by Alpha-Crystallin as a Mechanism of Preconditioning

“…3) in DPA was seen at 1% ethanol with freshly prepared GAPDH. We know that storage of proteins contributes to the lability of protein structure and function [30], likely making GAPDH more susceptible to DPA in the presence of ethanol. This susceptibility may be due to ethanol's greater access to the surface accessible cavities in stored proteins.…”

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles

“…The soluble fraction was used to purify the fusion protein. Recombinant proteins were purified by immobilized metal affinity chromatography using a Ni-chelating resin (Qiagen, Valencia, CA, USA) and eluted with 250 mM imidazole in a sodium phosphate buffer, as previously reported [ 40 ].…”