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Expression of active human β-glucuronidase in Sf9 cells infected with recombinant baculovirus
- Source :
-
Life Sciences . Aug2002, Vol. 71 Issue 13, p1547. 11p. - Publication Year :
- 2002
-
Abstract
- Antibody directed enzyme prodrug therapy (ADEPT) using glucuronide prodrugs is an experimental approach to reduce systemic toxicity of anti-cancer agents. Bioactivation of such prodrugs is achieved by fusion proteins consisting of targeting moieties (e.g. ligands of tumor specific antigens) and human β-glucuronidase. In order to test a large panel of possible β-glucuronidase fusion proteins for their applicability in ADEPT, an easy, rapid and high-yield expression system like the baculovirus/insect cell expression system would be needed. A prerequisite for using such fusion proteins is functional and biochemical characterization of human β-glucuronidase expressed in baculovirus-infected insect cells. Therefore, recombinant human β-glucuronidase was expressed in Sf9 insect cells and characterized at the protein and functional level. As shown by Western blot analysis the recombinant enzyme consists of dimers with their monomers being linked via disulfide bonds. Posttranslational modifications of the monomers seem to be different as compared with mammalian cells or tissues. The enzyme is functionally active in cleaving the substrates 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid, 4-methylumbelliferyl-β-D-glucuronide and the glucuronide prodrug HMR 1826, respectively, with similar enzyme kinetic parameters as those found in human tissues. Our data demonstrate that β-glucuronidase expressed in Sf9 cells displays the same enzymatic features as the protein expressed in mammalian cells. Therefore, we suggest that β-glucuronidase fusion proteins produced in this cell line will be valuable tools for testing a large panel of various targeting moieties in human tumor xenograft models or may be used for ADEPT in man. [Copyright &y& Elsevier]
- Subjects :
- *PRODRUGS
*ANTINEOPLASTIC agents
*BACULOVIRUSES
Subjects
Details
- Language :
- English
- ISSN :
- 00243205
- Volume :
- 71
- Issue :
- 13
- Database :
- Academic Search Index
- Journal :
- Life Sciences
- Publication Type :
- Academic Journal
- Accession number :
- 7846104
- Full Text :
- https://doi.org/10.1016/S0024-3205(02)01917-3