Author/Editor		Urbič, Tjaša
Title		Priprava rekombinantne človeške lizosomske dipeptidaze
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2004
Volume		str. 53
Language		slo
Abstract		Lysosomal dipeptidase (LDP) is a metallopeptidase belonging to the M28 family of the MH clan: 1f requires two zinc ions for its catalytic activity. Lysosomal dipeptidase was expressed as a proenzyme using the Escherichia coli and Pichia pasforis expression systems. For efficient expression and purification, we inserted the cDNA corresponding to the LDP proenzyme into a bacterial expression vector pET22. Expression resulted in the formation of insoluble cytoplasmic protein aggregates in the form of inclusion bodies. Protein prepared in this way was used to produce polyclonal antibodies against LDP. Due to the time consuming renaturation and low yield of the correctly folded protein produced in E. coli an alternative expression system was used. To obtain soluble protein we expressed LDP proenzyme in the P. pastoris expression system. The nucleotide sequence encoding LDP was cloned into the pGEM-T Easy clonal vector and then subcloned into the pGAPZa expression vector. Protein was expressed into the medium and purified by ion exchange chromatography on QSepharose Fast Flow and gel filtration chromatography on Sepharose S-200. The molecular mass of the recombinant LDP determined by gel filtration chromatography was estimated to be 120 kDa. Electrophoresis in the presence of sodium dodecyl sulphate (SDS) and Western blot analysis resulted in a band at 60 kDa. Based on these results we conclude that recombinant lysosomal dipeptidase is a homodimer, as is natural LDP. Pepsin and cathepsins C, D and L were used in an attempt to produce a catalytically active enryme. Lysosomal dipeptidase activity was measured by the hydrolysis of the substrate Ser-Met. The activation succeeded with pepsin in the presence of zinc ions and pH between 4.0 and 4.5.
Descriptors		DIPEPTIDASES LYSOSOMES RECOMBINANT PROTEINS TRANSFORMATION, GENETIC BASE SEQUENCE POLYMERASE CHAIN REACTION CHROMATOGRAPHY, ION EXCHANGE CHROMATOGRAPHY, GEL ESCHERICHIA COLI PICHIA BLOTTING, WESTERN MOLECULAR WEIGHT