Avtor/Urednik		Fonovič, Marko
Naslov		Izražanje in izolacija rekombinantnih človeških katepsinov O in F
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		2001
Obseg		str. 76
Jezik		slo
Abstrakt		Recombinant human cathepsins O and F were obtained from geneticaly modified bacterial and yeast cells. Yeast P. pastories, with human cathepsin F DNA inserted in its genome (yeast strain was provided by dr. Dieter Bromme, Mount Sinai School of Medicine, New York) was grown in fermentor. Expression of cathepsin F was induced by addition of methanol in the growth medium. Recombinant cathepsin F was isolated from fermentation broth with hydrophobic and gel chromatography. Protein identity was confirmed by immunological detection, enzyme activity measurement and N-terminal aminoacid analysis. Protein purity and homogenity was checked by SDS-PAGE and PAGE under native conditions. Isolation of cathepsin F was very troublesome and it was suspected that this is due to overglycosilation of the protein. In order to avoid this problem a mutant of cathepsin F cDNA without N-glycosilation sites, was prepared. Desired mutations were confirmed by DNA sequence analysis. Mutant cDNA cloned in expression plasmids pPIC9 and pGAPZalfa and yeast P. pastoris was transformed with both plasmids. After test expression no mutant cathepsin F was detected in the growth medium. No mutant protein was expressed in yeast cells. Procathepsin O cDNA was inserted in expression plasmid pGAPZalfa and P.pastoris strain X-33 was transformed by constructed plasmid. Transformed yeast cells were grown in shaker flasks and protein expression was controled by SDS-PAGE. After 4-6 days of expression a protein band with molecular mass similar to procathepsin O, was observed. Presence of procathepsin O was confirmed with N-terminal aminoacid sequence analysis. Procathepsin O was partially purified by gel chromatography. Activation at acidic pH was performed and monitored by SDS-PAGE. (Abstract truncated at 2000 characters).
Deskriptorji		CATHEPSINS PICHIA ESCHERICHIA COLI RECOMBINANT PROTEINS TRANSFORMATION, GENETIC PLASMIDS DNA POLYMERASE CHAIN REACTION SEQUENCE ANALYSIS, DNA AMINO ACID SEQUENCE