Abstract
Recombinant human Glutaminyl Cyclase expressed in E. coli 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.
Highlights
Glutaminyl-peptide Cyclotransferase (QPCT), known as Glutaminyl Cyclase (QC), catalyzes the conversion of N-terminal L-glutaminyl peptide residues to pyroglutamyl groups, a process required for the maturation of numerous bioactive peptides [1]
Murine and human proteins recombinantly expressed in the yeast Pichia pastoris are instead glycosylated and their X-ray structure has revealed some loop rearrangements in the neighborhood of the active center [5], the extent of these rearrangements being smaller for hQPCT
To what reported for the expression of hQPCT in pET vectors [5], from our expression trials the best condition turned out to be 17uC, 0.2 mM IPTG for 48 hours in rich medium using BL21DE3 as E. coli strain
Summary
Glutaminyl-peptide Cyclotransferase (QPCT), known as Glutaminyl Cyclase (QC), catalyzes the conversion of N-terminal L-glutaminyl peptide residues to pyroglutamyl groups, a process required for the maturation of numerous bioactive peptides [1]. From the analysis of the electrostatic surface generated by PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrodinger, LLC) together with the evaluation of the atomic accessible surface performed by Naccess [24] (see Materials and Methods), two main hydrophobic solvent exposed patches close to the active site and probably responsible for the hQPCT aggregation, were identified (Fig. 4A and 4B ).
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