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Expression and characterization of the multidrug resistance-associated protein in insect cells infected with a
H Sun1, Q Zhu, M S Center
1Division of Biology, Kansas State University, Manhattan 66506, USA.
Abstract:
The protein encoded by the multidrug resistance-associated protein (MRP) gene was examined after infection of SF21 insect cells with recombinant baculovirus containing a full-length MRP cDNA. The time course of appearance of the protein as determined by western blot analysis revealed that maximum levels occurred 2 days postinfection. The amount of MRP made in this system was somewhat variable, but levels that were about 4-fold greater than that found in HL60/ADR cells could be achieved. The protein appeared to be full-length but was present in a highly deglycosylated form. The P170 (MRP) was phosphorylated and located exclusively in membranes of infected cells. P170 (MRP) synthesized in this system was capable of carrying out the ATP-dependent transport of leukotriene C4 into isolated membrane vesicles. The results thus indicate that MRP synthesized in insect cells is functional and has properties similar to the authentic protein found overexpressed in certain multidrug-resistant isolates.
Insights
Researchers produced functional multidrug resistance-associated protein (MRP) in insect cells. This synthesized MRP protein exhibited properties similar to the authentic protein found in multidrug-resistant cells.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- The multidrug resistance-associated protein (MRP) is implicated in multidrug resistance.
- Understanding MRP protein function and production is crucial for developing resistance-modulating therapies.
Purpose of the Study:
- To investigate the production and functionality of the multidrug resistance-associated protein (MRP) in insect cells using a recombinant baculovirus system.
- To characterize the properties of MRP synthesized in an insect cell expression system.
Main Methods:
- Infection of SF21 insect cells with recombinant baculovirus containing full-length MRP cDNA.
- Western blot analysis to determine protein expression levels and time course.
- Analysis of protein glycosylation, phosphorylation, and cellular localization.
- Functional assay of ATP-dependent transport of leukotriene C4 into isolated membrane vesicles.
Main Results:
- Maximum MRP protein levels were achieved 2 days post-infection, with levels up to 4-fold higher than in HL60/ADR cells.
- The synthesized MRP protein was full-length but highly deglycosylated.
- Phosphorylated MRP was localized exclusively in the membranes of infected cells.
- Insect cell-synthesized MRP demonstrated ATP-dependent transport of leukotriene C4, indicating functionality.
Conclusions:
- Insect cells can synthesize functional multidrug resistance-associated protein (MRP).
- The synthesized MRP protein exhibits properties comparable to the authentic MRP found in multidrug-resistant cells.
- This insect cell expression system provides a viable method for studying MRP function and potentially for producing the protein.