Expression and purification of human stromelysin 1 and 3 from baculovirus-infected insect cells.
Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 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 remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression 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 alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including 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 obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies.
Departamento de Investigación, Pharmacia & Upjohn, Antonio López 109, Madrid, 28026, Spain.
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del Mar Barbacid
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Fernández-Resa
J M
JM
Buesa
G
G
Márquez
M
M
Aracil
A R
AR
Quesadaand
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E
Mira
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D053507:Matrix Metalloproteinase 11; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D000511:alpha-Macroglobulins / Q000378:metabolism
D002364:Caseins; D018076:DNA, Complementary; D011498:Protein Precursors; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins; D053507:Matrix Metalloproteinase 11; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3