Characterisation of human dipeptidyl peptidase IV expressed in Pichia pastoris.: A structural and mechanistic comparison between the recombinant human and the purified porcine enzyme

Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the Nterminus 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 (K-d=153.0 +/- 17.0 M and K-d=33.4 +/- 2.2 muM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (K-d=2.15 +/- 0.18 nM and K-d=7.38 +/- 0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RTPCR, revealed 88% identity between both enzymes, the speciesspecific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.