One-step affinity purification of Bacillus neutral proteases using bacitracin-silica

Journal of Biochemical and Biophysical Methods
B Van den BurgG Venema

Abstract

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.

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Citations

Aug 1, 1993·Journal of Computer-aided Molecular Design·G Vriend, V Eijsink
Mar 15, 1994·European Journal of Biochemistry·B Van den BurgV G Eijsink
Aug 28, 2012·Applied and Environmental Microbiology·Laxmi KrishnappaJan Maarten van Dijl
Apr 10, 1995·FEBS Letters·A FontanaP Polverino de Laureto
Jan 12, 2005·Protein Expression and Purification·Johanna MansfeldRenate Ulbrich-Hofmann
Feb 23, 2002·The Journal of Biological Chemistry·Arno de KreijJens E Nielsen
Apr 25, 1997·The Journal of Biological Chemistry·J MansfeldV G Eijsink
Dec 18, 1998·The Journal of Biological Chemistry·G VriendV G Eijsink
Jan 1, 1991·Applied Biochemistry and Biotechnology·J S Dordick
Jul 8, 1991·FEBS Letters·S PfefferC Betzel
Sep 20, 1996·Journal of Chromatography. B, Biomedical Applications·O Ibrahim-Granet, O Bertrand

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