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Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation

Kilbane, Zelda and Vaas, Paul-Roman and Ó Cuiv, Padraig and O'Connor, Brendan (2007) Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli : purification , biochemical and kinetic characterisation. Molecular and Cellular Biochemistry, 297 (1-2). pp. 189-197. ISSN 0300-8177

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Abstract

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl-aminopeptidase I. The amino acid sequence, deduced from the nucleotide sequence, revealed that it consists of 209 amino acid residues and showed to have 98% homology with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary among the two sequences. The bovine enzyme was expressed in XL10-gold Esherichia coli cells. Immobilizied Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 3.3mg of PAP1 per litre culture. The purified enzyme had a specific activity of 1700 units/ml. SDS-PAGE produced a single band for bovine PAP1 with a molecular weight of ~23-24 kDa which is in good agreement with previously reported data on PAP1. Kinetic constants Km and Kcat were 59μΜ and 3.5s-1, respectively. It possessed an optimum pH between 9-9.5, a temperature of 37°C and showed an absolute requirement for a thiol-reducing agent (10mM DTT). EDTA didn’t prove to have an effect on enzyme activity. Competitive inhibition was seen with pyroglutamyl peptides pGlu-His-Pro-NH2 (TRH; Ki= 44.1 uM), pGlu-Ala- OH (Ki=141 uM) and pGlu-Val-OH (Ki=652.17).

Item Type:Article (Published)
Refereed:Yes
Uncontrolled Keywords:pyroglutamyl peptidase type-1; cloning; expression in E. coli ; purification; characterisation
Subjects:Biological Sciences > Biochemistry
Biological Sciences > Enzymology
Biological Sciences > Molecular biology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > Irish Separation Science Cluster (ISSC)
Publisher:Kluwer
Official URL:http://dx.doi.org/10.1007/s11010-006-9346-9
Copyright Information:© 2007 Springer (Kluwer) The original publication is available at www.springerlink.com
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
Funders:Enterprise Ireland
ID Code:17806
Deposited On:28 Feb 2013 14:20 by Brendan O'Connor. Last Modified 11 Oct 2017 13:08

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