PMID: 8612629Mar 1, 1996Paper

Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli

European Journal of Biochemistry
G KopitarV Turk

Abstract

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.

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Jul 1, 1992·Biological Chemistry Hoppe-Seyler·I DolencV Turk
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Citations

Jun 29, 1999·Biological Chemistry·V StokaV Turk
Jul 31, 2003·Biological Chemistry·Boris Turk, Hans Fritz
Aug 21, 2012·Dental Materials : Official Publication of the Academy of Dental Materials·Leo TjäderhaneDavid H Pashley
Mar 30, 2007·Protein Expression and Purification·Gerd KramerKlaus Schilling
Jun 6, 2003·Biochemical and Biophysical Research Communications·A OhashiN Katunuma
Feb 23, 2002·Protein Expression and Purification·Dominique A TobbellW Mark Abbott
Mar 21, 2015·World Journal of Microbiology & Biotechnology·Chunfang LingAilin Tao
Jan 15, 2015·BioMed Research International·Marko NovinecBoris Turk
Mar 21, 2001·Biotechnology & Genetic Engineering Reviews·L M BabéB F Schmidt
Apr 23, 2008·Current Protocols in Protein Science·D Brömme
Sep 17, 1998·The Journal of Biological Chemistry·G LalmanachF Gauthier
Nov 19, 2003·Biochemistry·Olga VasiljevaBoris Turk
Feb 16, 2000·Biochemical and Biophysical Research Communications·N KatunumaT Asao
Aug 24, 2013·Biochemistry·Juliette SageFabien Lecaille

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