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Purification and reconstitution of human P-glycoprotein
S V Ambudkar1, I H Lelong, J Zhang
1Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Methods in Enzymology
|August 26, 1998
Summary
This study details the purification of active human P-glycoprotein (Pgp), a key multidrug transporter, using chromatography. The purified Pgp retains its drug-transporting function, crucial for understanding multidrug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- P-glycoprotein (Pgp) is a crucial efflux pump involved in multidrug resistance (MDR).
- Efficient purification of active Pgp is essential for studying its function and developing MDR therapies.
Purpose of the Study:
- To establish a robust protocol for purifying biologically active human Pgp.
- To investigate the functional activity of purified Pgp as an ATP-dependent drug transporter.
Main Methods:
- Sequential chromatography using DEAE-sepharose CL-6B and wheat germ agglutinin (WGA) columns.
- Inclusion of glycerol and lipid during purification to maintain Pgp activity.
- Assay of drug-stimulated ATP hydrolysis and ATP-dependent vinblastine transport in proteoliposomes.
Main Results:
- A purification protocol was developed yielding stable and active human Pgp from cell lines and transfectants.
- Purified Pgp exhibited high levels of drug-stimulated ATP hydrolysis.
- Reconstitution of purified Pgp into proteoliposomes demonstrated ATP-dependent vinblastine transport.
Conclusions:
- The established purification method effectively retains Pgp biological activity.
- Purified Pgp functions as an ATP-dependent multidrug transporter, validating its role in drug efflux.