Real-time kinetic analysis of limited proteolysis by ion-exchange chromatography using compressed, non-porous agarose beads.
In biotechnology there is a great need for methods that can be used for kinetic studies of ongoing processes, such as limited proteolysis. Ion-exchange chromatography on a nonporous, compressed agarose matrix was used in the study presented in this paper to monitor papain cleavage of fungal cellulases. This non-porous matrix allows completion of a chromatographic analysis, with baseline resolution, within 30-60 s. Utilizing this very fast chromatographic method one can monitor a proteolysis 'continuously' by repeated chromatographic analysis of the incubation mixture and, if desired, terminate it at the optimal moment. Fractions were also collected for identification by activity assays and analytical SDS-PAGE.
Mohammad
J
J
Department of Biochemistry, University of Uppsala, Sweden.
Eriksson
K O
KO
Johansson
G
G
D002480:Cellulase / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003199:Computer Systems; D005658:Fungi / Q000201:enzymology; D007700:Kinetics; D008863:Microspheres; D010206:Papain; D016062:Porosity; D012685:Sepharose; D014242:Trichoderma / Q000201:enzymology
D012685:Sepharose; D002480:Cellulase; D010206:Papain