Soluble Variants of Human Recombinant Glutaminyl Cyclase

<div><p>Recombinant human Glutaminyl Cyclase expressed in <i>E. coli</i> is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of ¹H-¹⁵N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease.</p></div

NMR spectra of wild type, 2xmut and 6xmut.

<p>2D ¹H-¹⁵N-HSQC spectra of: <b>A,</b> 30 µM wild type hQPCT at 700 MHz; <b>B,</b> of 90 µM 2xmut hQPCT at 950 MHz; <b>C,</b> 70 µM 6xmut hQPCT at 950 MHz. Spectra were recorded at 298 K, in 150 mM NaCl and 50 mM Tris pH 8 buffer.</p

3D structure of hQPCT.

<p>Ribbon representation of the X-ray structure of hQPCT (PDB id 2AFM). The zinc ion is shown by a yellow sphere, the zinc ligands are shown as orange sticks and the two Cys residues responsible for the disulphide bridge formation as green sticks. The loop connecting β1 with β2 is highlighted in red, while those forming the crown-like structure around the zinc are in orange.</p

Surface charge representation of hQPCT, single mutants and residues used as input for docking calculation.

<p><b>A,</b> Surface charge representation of hQPCT (PDB id 2AFM) where region of positive, negative and neutral electrostatic potential are indicated in blue, red and white, respectively. The electrostatic surfaces were generated using the software PyMOL with the command “generate vacuum electrostatic”. The protein orientation is the same as in Fig. 1. The ribbon representation of the protein is visible in transparency. Amino acids belonging to the hydrophobic regions 1 and 2 used as input for docking calculation are clustered into two groups and shown as cyan and green sticks, respectively. Single point mutations I47, L289, F260 are highlighted as orange spheres. The zinc ion is represented as a yellow sphere. <b>B,</b> Different views of hQPCT that allow visualizing the location of the hydrophobic regions 1 and 2 on the protein surface.</p

Purification of wild type hQPCT.

<p><b>A,</b> Imidazole gradient (green line) (a) 50 mM, (b) 50–500 mM, (c) 500 mM in FPLC Akta (GE Healthcare). Blue line: UV measure (mAU). <b>B,</b> SDS-PAGE of purified protein fractions. Lane 1: insoluble fraction, lane 2: protein marker, lane 3: total fraction, lane 4: flow-through, lane 5: wash unbound, lanes 6–7: fractions 50 mM imidazole, lanes 8–15: fractions 50–500 mM imidazole.</p

Purification of 2xmut hQPCT.

<p><b>A,</b> Ni-NTA affinity purification-imidazole gradient (green line: a, 50 mM; b, 50–500 mM) in FPLC Akta (GE Healthcare). Blue line: UV measure (mAU). <b>B,</b> SDS-PAGE gel of imidazole gradient fractions, lane 1: insoluble fraction, lane 2: protein marker, lane 3: total fraction, lane 4: flow-through, lane 5: wash unbound, lanes 7–15: fractions 50–500 mM imidazole. <b>C,</b> Size Exclusion in HiLoad 16/60 Superdex 75 column in FPLC Akta (GE Healthcare). Blue line: UV measure (mAU). <b>D,</b> SDS-PAGE, lane 1: protein marker, lanes 6–12: monomeric 2xmut hQPCT.</p