EXPRESSION AND PURIFICATION OF HUMAN STROMELYSIN-1 AND STROMELYSIN-3 FROM BACULOVIRUS-INFECTED INSECT CELLS

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromely sin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression rem ains to be elucidated. Genes encoding ST1 and ST3 were expressed in le pidopteran insect cells using the baculovirus expression vector system . Recombinant baculoviruses were obtained after cloning the full-lengt h cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpres sed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expres sion being reached from 24 h postinfection, After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha 2-macroglobulin, casein, fibronectin, alpha 1-antitrypsin, a nd laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h po stinfection. After purification to 95% homogeneity, 2.5 mg of proST3 w as obtained per 5 x 10(8) infected cells. A number of proteases includ ing plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtain ed protein, 2.5 mg per 5 x 10(8) infected cells purified to 80% homoge neity, was shown to be active on both casein degradation and alpha 2-m acro-globulin entrapment assays. Our results suggest that the baculovi rus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies. (C) 1998 Academ ic Press.