Recombinant human mast cell tryptase : stable expression in Pichia pastoris and purification of fully active enzyme

• Published in: Biotechnology and applied biochemistry 1998-10, Vol.28 (2), p.125-131
• Main Authors: NILES, A. L, MAFFITT, M, HAAK-FRENDSCHO, M, WHEELESS, C. J, JOHNSON, D. A
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Enzyme engineering; Fermentation; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Production of selected enzymes
• ISSN: 0885-4513; 1470-8744

Human mast cell tryptase (EC 3.4.21.59) is a trypsinlike serine protease that is stored in and released from mast cell granules. This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome. Cells producing recombinant human tryptase 1 (rHT) were selected by screening with antibodies. Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium. Increasing levels of enzyme were detected in the medium for up to 3 days. Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two-step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE. These multiple forms were due to differences in post-translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa. A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration. Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar K sub(m) values, but the k sub(cat) of rHT was more than twice that of HLT.