Avtor/Urednik		Kopitar, G; Dolinar, M; Štrukelj, B; Pungerčar, J; Turk, V
Naslov		Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli
Tip		članek
Vir		Eur J Biochem
Vol. in št.		Letnik 236, št. 2
Leto izdaje		1996
Obseg		str. 558-62
Jezik		eng
Abstrakt		Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro-5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth.
Deskriptorji		CATHEPSINS ENZYME PRECURSORS PROTEIN FOLDING BASE SEQUENCE DNA PRIMERS ENZYME ACTIVATION ESCHERICHIA COLI MOLECULAR SEQUENCE DATA RECOMBINANT PROTEINS