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• W1649005596 abstract "Prolidase is a Mn2+-dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 °C was therefore undertaken. For this analysis, an ‘on-column’ digestion of the N-terminal His tag by Factor Xa was performed. A positive effect of Mn2+ and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy." @default.
• W1649005596 created "2016-06-24" @default.
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• W1649005596 date "2006-12-01" @default.
• W1649005596 modified "2023-09-25" @default.
• W1649005596 title "Human recombinant prolidase from eukaryotic and prokaryotic sources." @default.
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• W1649005596 doi "https://doi.org/10.1111/j.1742-4658.2006.05538.x" @default.
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