caa a22 4500 378855964 CHVBK 20180305123335.0 cr unu---uuuuu 161128e20031225xx s 000 0 eng 10.1515/BC.2003.172 doi (NATIONALLICENCE)gruyter-10.1515/BC.2003.172 Characterisation of Human Dipeptidyl Peptidase IV Expressed in Pichia pastoris. A Structural and Mechanistic Comparison between the Recombinant Human and the Purified Porcine Enzyme [Elektronische Daten] [J. Bär, A. Weber, T. Hoffmann, J. Stork, M. Wermann, L. Wagner, S. Aust, B. Gerhartz, H.-U. Demuth] Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DPIV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-17 -36 resulted in significantly different binding constants for the human and the porcine enzyme (Kd=153.0±17.0 M and Kd=33.4± 2.2 uM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd=2.15±0.18 nM and Kd=7.38±0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding. Copyright © 2003 by Walter de Gruyter GmbH & Co. KG Biochemistry nationallicence Molecular biology nationallicence Cellular biology nationallicence Bär J. aut Weber A. aut Hoffmann T. aut Stork J. aut Wermann M. aut Wagner L. aut Aust S. aut Gerhartz B. aut Demuth H.-U aut Biological Chemistry Walter de Gruyter 384/12(2003-12-25), 1553-1563 1431-6730 384:12<1553 2003 384 bchm https://doi.org/10.1515/BC.2003.172 text/html Onlinezugriff via DOI 1 research article jats NATIONALLICENCE 856 40 https://doi.org/10.1515/BC.2003.172 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Bär J. aut NATIONALLICENCE 700 1- Weber A. aut NATIONALLICENCE 700 1- Hoffmann T. aut NATIONALLICENCE 700 1- Stork J. aut NATIONALLICENCE 700 1- Wermann M. aut NATIONALLICENCE 700 1- Wagner L. aut NATIONALLICENCE 700 1- Aust S. aut NATIONALLICENCE 700 1- Gerhartz B. aut NATIONALLICENCE 700 1- Demuth H.-U aut NATIONALLICENCE 773 0- Biological Chemistry Walter de Gruyter 384/12(2003-12-25), 1553-1563 1431-6730 384:12<1553 2003 384 bchm CC0 http://creativecommons.org/publicdomain/zero/1.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-gruyter