Recombinant Human Mast-Cell Chymase: An Improved Procedure for Expression in Pichia Pastoris and Purification of the Highly Active Enzyme
Document Type
Article
Publication Date
2-1-2005
Description
Human mast-cell chymase (EC 3.4.21.39) is a chymotrypsin-like serine protease that is stored in and released from mast-cell granules. This enzyme has been expressed in Pichia pastoris by homologous recombination of the cDNA coding for the mature active chymase into the Pichia genome. Cells producing the highest levels of recombinant human chymase were selected by activity screening and they were grown in a fermentor. Methanol induction resulted in the secretion of active chymase into the Pichia growth media and increasing levels of enzyme were detected in the media for 5 days. Active enzyme was purified from the culture media with a 22 % yield of activity by a simple two-step procedure involving hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media.
Citation Information
Lockhart, Brent E.; Vencill, Jessica R.; Felix, Cherise M.; and Johnson, David A.. 2005. Recombinant Human Mast-Cell Chymase: An Improved Procedure for Expression in Pichia Pastoris and Purification of the Highly Active Enzyme. Biotechnology and Applied Biochemistry. Vol.41(1). 89-95. https://doi.org/10.1042/BA20040074 PMID: 15163313 ISSN: 0885-4513