Characterization of recombinant human cathepsin B expressed at high levels in baculovirus.
The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper-glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus-expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.
Steed
P M
PM
Research Department, Novartis Pharmaceuticals, Summit, New Jersey 07901, USA. paul.steed@pharma.novartis.com
Lasala
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D
Liebman
J
J
Wigg
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Clark
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Knap
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AK
D016367:Baculoviridae / Q000235:genetics; D002401:Cathepsin B / Q000737:chemistry*; D003001:Cloning, Molecular; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007700:Kinetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry*
D011994:Recombinant Proteins; D002401:Cathepsin B