Soluble Variants of Human Recombinant Glutaminyl Cyclase

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.

The soluble Y115E–Y117E variant of human glutaminyl cyclase is a valid target for X-ray and NMR screening of inhibitors against Alzheimer disease

“…The supernatant of the resulting crude extract was collected by centrifugation and further purified by nickel-affinity chromatography similar to the previously described method (Castaldo et al, 2013).…”

“…Glutaminyl cyclase (QC) belongs to the class of transferases and catalyzes the formation of pyroglutamic acid (pGlu) from N-terminal glutaminyl or glutamyl precursors of several bioactive peptides and proteins. QC has been identified in animals, plants and bacterial sources (Castaldo et al, 2013;Busby et al, 1987;Messer, 1963). Owing to its potential involvement in the formation of pGlumodified amyloid peptides, hQC is considered to be a valid drug target in Alzheimer disease (AD) and in the familial British and Danish dementias (Schilling et al, 2008).…”

“…Consequently, a general method that is able to provide milligrams of soluble hQC is of great practical importance (Baneyx, 1999). A soluble variant of hQC has been designed in order to overcome the tendency of recombinant bacterial hQC to be expressed as inclusion bodies (Castaldo et al, 2013). This goal has been reached by substituting two tyrosine residues on the surface of hQC with glutamates to obtain a soluble but still active form of the protein (Castaldo et al, 2013).…”