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

Kilbane, Zelda, Vaas, Paul-Roman, Ó Cuív, Páraic 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: 189-197. doi:10.1007/s11010-006-9346-9


Author Kilbane, Zelda
Vaas, Paul-Roman
Ó Cuív, Páraic
O'Connor, Brendan
Title Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation
Formatted title
Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation
Journal name Molecular and Cellular Biochemistry   Check publisher's open access policy
ISSN 0300-8177
1573-4919
Publication date 2007-03
Sub-type Article (original research)
DOI 10.1007/s11010-006-9346-9
Open Access Status
Volume 297
Issue 1-2
Start page 189
End page 197
Total pages 9
Place of publication New York, NY, United States
Publisher Springer New York
Language eng
Formatted abstract
We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His6 tag (rBtaPAP16H) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP16H had a specific activity of 3633 units mg−1. SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of ~24 kDa, which is in good agreement with previously reported data on PAP1. The Km and kcat values obtained for rBtaPAP16H were 59 μM and 3.5 s−1, respectively. The optimum pH for activity was 9.0–9.5 and the optimum temperature was 37 °C. rBtaPAP16H was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH2 (TRH), pGlu-Ala and pGlu-Val revealed Ki values of 44.1, 141 and 652.17 μM, respectively. The lowest Ki, observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP16H has a higher affinity for tripeptides over dipeptides.
Keyword Pyroglutamyl peptidase type-1
Cloning
Expression in E. coli
Purification
Characterisation
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: UQ Diamantina Institute Publications
 
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Created: Wed, 19 Mar 2014, 11:34:30 EST by Paraic O Cuiv on behalf of UQ Diamantina Institute