Recombinant human mast-cell chymase: an improved procedure for expression in Pichia pastoris and purification of the highly active enzyme.
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.
Lockhart
Brent E
BE
Department of Biochemistry and Molecular Biology, J.H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0581, USA.
Vencill
Jessica R
JR
Felix
Cherise M
CM
Johnson
David A
DA
D018929:Cell Culture Techniques / Q000379:methods*; D053818:Chymases; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D008407:Mast Cells / Q000201:enzymology*; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D053818:Chymases