Author/Editor		Fonović, Marko
Title		Izražanje, izolacija in delna karakterizacija rekombinantnih človeških katepsinov O in F
Type		monografija
Place		Ljubljana
Publisher		Univerza v Ljubljani, Medicinska fakulteta
Publication year		2004
Volume		str. 110
Language		slo
Abstract		Human procathepsins F and O were expressed in Sf9 insect cell line. Recombinant procathepsin F was purified from the media and processed to the mature form. Kinetic parameters of its interaction with various fluorogenic substrates and endogenous protein inhibitors werc determined. Cathepsin F hydrolysed Z-FR-AMC much faster than Z-FVRAMC, while hydrolysis of Z-RR-AMC was neglectable. However, cathepsin F expressed in the insect cells hydrolysed Z-FR-AMC less efficiently than its counterpart produced in yeast. This is probably due to the difference in glycosylation because insect cells are capable of producing more complex forms of oligosaccharide chains. The inhibition constants for interaction of cathepsin F with endogenous protein inhibitors were in picomolar and nanomolar range, which is characteristic for the inhibition of endoproteases. The strongest and the fastest inhibition was observed for cystatin C and chicken cystatin, while inhibition by stefin A was the weakest, which is unusual for the papain-like cathepsins. When expressed in HEK-293 cell line, preprocathepsin F-GFP construct was observed in vesicles, which is common for proteases of papain family. Recombinant procathepsin O purified from insect cells could not be activated autocatalyticaly or by the action of other proteases. The hypothesis that activation of procathepsin O is hindered by glycosylation (a putative glycosylation site is located next to the proregion cleavage site) has been tested. We prepared a mutant cDNA of procathepsin O with deleted glycosylation site and expressed it in the Sf9 insect cell line. Mutant procathepsin O appeared to be activated during expression but it could not be purified and characterized because of the problems with precipitation during the purification procedure. Preprocathepsin O-GFP construct expressed in HEK-293 cell line, was observed in vesicles (similar as procathepsin F). (Abstract truncated at 2000 characters).
Descriptors		CATHEPSINS RECOMBINANT PROTEINS PLASMIDS TRANSFORMATION, GENETIC ESCHERICHIA COLI INSECTS TRANSFECTION CELLS, CULTURED POINT MUTATION SEQUENCE ANALYSIS, DNA POLYMERASE CHAIN REACTION