Production, purification, and characterization of recombinant maspin proteins.
Maspin, a novel mammary serine protease inhibitor, was shown to have tumor suppressing activity (Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E., and Sager, R. (1994) Science 263, 526-529). In this paper, we report the production of recombinant glutathione S-transferase-maspin fusion protein, expressed in the bacterium Escherichia coli, and recombinant maspin, expressed in the insect Spodoptera frugiperda cells. The fusion protein was purified by glutathione affinity chromatography. Maspin expressed in insect cells was purified by a combination of Bio-Rad AG1-2X anion exchange chromatography and heparin affinity chromatography. The recombinant maspin from insect cells was cleaved at the putative reactive center, as confirmed by protein sequencing. Both recombinant proteins demonstrated strong inhibitory effects on the invasion by two breast tumor cell lines across reconstituted basement membranes and such inhibitory effect was abolished in the presence of the polyclonal antibody made against the reactive center region of maspin. The trypsin-cleaved recombinant maspin did not inhibit invasion, indicating that the inhibitory activity requires the intact putative reactive center. This paper provides evidence that recombinant maspin protein itself inhibits invasion, and supports the role of maspin as a tumor suppressor.
Sheng
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S
Division of Cancer Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Pemberton
P A
PA
Sager
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R
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000473:pathology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen; D003429:Cross Reactions; D004338:Drug Combinations; D016147:Genes, Tumor Suppressor; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007797:Laminin; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness / Q000517:prevention & control; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D015843:Serpins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera; D014357:Trypsin; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004338:Drug Combinations; D007797:Laminin; D011506:Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; C085344:SERPIN-B5; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C059485:matrigel; D003094:Collagen; D005982:Glutathione Transferase; D014357:Trypsin