Recombinant expression of human mast cell proteases chymase and tryptase.
Expression of recombinant human chymase and tryptase was achieved in a baculovirus-insect cell system using a fusion protein construct. Recombinant baculovirus was produced with DNA coding for a NH2-ubiquitin-chymase-COOH or NH2-ubiquitin-tryptase-COOH fusion protein inserted immediately downstream of the signal sequence for the secreted envelope protein, glycoprotein 67. In each construct, the natural prepropeptide sequence of the protease was replaced by the amino acid sequence for the enterokinase cleavage site of trypsinogen. High Five insect cells infected with either of the modified baculovirus produced mg quantities of each fusion protein per liter of culture. Treatment of the chymase-fusion protein with enterokinase or the tryptase-fusion protein with enterokinase in the presence of a highly charged polysaccharide (dextran sulfate or heparin) produced enzymatically active proteases with properties of the native enzymes. A procedure for the purification of mg quantities of recombinant chymase from infected-cell medium is presented.
Wang
Z
Z
Department of Medicine, University of Pennsylvania, Philadelphia 19104-6142, USA.
Walter
M
M
Selwood
T
T
Rubin
H
H
Schechter
N M
NM
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D053818:Chymases; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008407:Mast Cells / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D053802:Tryptases
D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases