PMID: 9172785Feb 1, 1996Paper

Expression and purification of recombinant human tryptase in a baculovirus system

Protein Expression and Purification
K SakaiLawrence B Schwartz

Abstract

B2 is a mAb that recognizes a conformational determinant on the active form of native tryptase, but does not recognize native tryptase that spontaneously loses activity in physiologic buffer. Precursor forms of recombinant human (rh) alpha- and rh beta-tryptase have been expressed in a baculovirus system. In each case, multiple electrophoretic forms were detected in both culture media and cell lysates of infected insect cells by Western blots developed with the G3 mAb made against native human tryptase. Although only 4 of 30 amino acids in the leader sequences of alpha- and beta-tryptase differ, rh alpha-tryptase appeared predominantly in the cell lysates, rh beta-tryptase predominantly in the culture media. B2 recognized rh alpha-tryptase and rh beta-tryptase found in the culture media of infected Sf-9 cells, but not that in cell lysates. Secreted forms of tryptase were purified to homogeneity by B2-immunoaffinity chromatography. From 1 liter of culture fluid 1.5 to 3 mg of rh-tryptase could be purified. Each rh-tryptase precursor was enzymatically inactive with synthetic substrates. Analysis of the N-terminal amino acid sequences of purified rh alpha- and rh beta-tryptase precursors (APAPVQA and APAPGQA, respectively) indicat...Continue Reading

Citations

Apr 2, 1998·Biological Chemistry·Z WangN M Schechter
Mar 30, 2007·Journal of Molecular Biology·Guido C PaesenDavid I Stuart
Apr 28, 2006·The FEBS Journal·Jenny Hallgren, Gunnar Pejler
Jul 26, 2000·Hematology/oncology Clinics of North America·L B Schwartz, A M Irani
Mar 27, 1999·Protein Expression and Purification·H ChanJ M Clark
Apr 15, 2004·Bioorganic & Medicinal Chemistry Letters·James C SuttonGregory S Bisacchi
May 13, 2011·The Journal of Immunology : Official Journal of the American Association of Immunologists·Quang T LeLawrence B Schwartz
Jun 8, 2017·Frontiers in Cellular and Infection Microbiology·Adrien A BlisnickSarah I Bonnet
Jul 12, 2011·The Journal of Immunology : Official Journal of the American Association of Immunologists·Quang T LeLawrence B Schwartz
May 22, 2003·The Journal of Immunology : Official Journal of the American Association of Immunologists·Lawrence B SchwartzYoshihiro Fukuoka
Mar 10, 2000·Biochimica Et Biophysica Acta·C P SommerhoffH Fritz

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