Expression and purification of recombinant human tryptase in a baculovirus system.
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) indicated that the initial 18 amino acids of the 30-amino-acid leader sequence had been removed. Differential N-glycosylation was found in both rh alpha-tryptase (one or two carbohydrate groups per molecule) and rh beta-tryptase (zero or one carbohydrate group per molecule). Thus, the baculovirus expression system is a useful tool for generation of rh alpha- and rh beta-tryptase precursors that exhibit a conformational epitope also present on natural tryptase and that are preferentially secreted into the culture media of infected cells.
Sakai
K
K
Division of Rheumatology, Allergy and Immunology, Department of Medicine, Commonwealth University, Richmond, Virgina 23298, USA.
Long
S D
SD
Pettit
D A
DA
Cabral
G A
GA
Schwartz
L B
LB
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D018411:Spodoptera; D053802:Tryptases
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase