Purification and partial characterization of recombinant human dipeptidyl-peptidase III

Ljudska dipeptidil-peptidaza III (DPP III), član porodice metalopeptidaza M49, enzim je s ulogom u intracelularnom katabolizmu proteina. S ciljem istraživanja uloge evolucijski sačuvanog ostatka Asn406, u ovom diplomskom radu su sintetizirani divlji tip DPP III i njen mutirani oblik N406S prekomjernom ekspresijom u bakteriji Escherichia coli transformiranoj plazmidom koji je sadržavao cDNA za ljudsku DPP III s privjeskom od 6 His. Nakon pročišćavanja afinitetnom kromatografijom, rekombinantnim proteinima je određena molekulska masa (81 kDa) SDS-elektroforezom, specifična aktivnost za hidrolizu kromogenog supstrata Arg-Arg-2-naftilamida (37,81 jmg-1 za divlji tip, 14,52 jmg-1 za mutant N406S), optimalni pH (8,6) i temperatura djelovanja (37 °C), te temperaturna stabilnost (do 37 °C za divlji tip, do 30 °C za N406S). Ustanovljena je inhibicija obaju enzimskih oblika kelatorima metala i sulfhidrilnim reagensom p-hidroksimerkuribenzoatom, te selektivnost za supstrat Arg-Arg-2-naftilamid. Rezultati ukazuju na važnost Asn406 za postizanje pune katalitičke aktivnosti i za termičku stabilnost ljudske DPP III.Human dipeptidyl-peptidase III (DPP III) is a member of the metallopeptidase family M49 with implied role in intracellular protein catabolism. To investigate the role of highly conserved Asn406, herein the wild-type DPP III and mutant N406S were overexpressed in Escherichia coli transformed with plasmid containing the cDNA encoding His6-tagged human DPP III. Recombinant proteins were purified by affinity chromatography, and their molecular mass (81 kDa) was determined by SDS-PAGE, as well as the specific activity for hydrolysis of chromogenic substrate Arg- Arg-2-naphthylamide (37.81 Umg-1, wild-type; 14.52 Umg-1, mutant N406S), pH and temperature optima (at 8.6, respectively 37 °C), and thermal stability (up to 37 °C for the wild-type, up to 30 °C for N406S). Both enzymes were inhibited by metallochelators and p-hydroxymercuri-benzoate, and showed selectivity for Arg-Arg-2-naphthylamide. The results suggest that Asn406 could be important for acquirement of full catalytic activity, and may contribute to human DPP III thermal stability