Ciblage de molécules vers des cellules eucaryotes à l'aide d'un transporteur protéique construit par ingénierie de la glutathion S-transférase P1-1 humaine
(2003)
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- Abstract
- The purpose of this project is to improve the efficiency of drug targeting to mammalian cells by coupling a drug to a protein carrier which delivers it specifically to the cells. The carrier is constructed by modifying two regions of the structure of a human enzyme. The active site is modified to facilitate drug conjugation and a binding site for a receptor expressed on the surface of the target cells is engineered in a flexible region of the protein located at distance from the active site to enable the recognition of the drug-carrier complex. The protein to be modified into hybrid carrier is the human glutathione S-transferase P1-1 (GSTP1-1) which plays a key role in the detoxification of a large variety of hydrophobic xenobiotics by catalysing the nucleophilic attack of the xenobiotic by the glutathione. This enzyme is active as a homodimer (46 kDa) encoded by a single gene. Each monomer is built of 209 residues and features a highly specific site for glutathione (G-site) and a poorly specific one for the xenobiotics (H-site). On the one hand, as the drug conjugation to the carrier occurs through a disulfide bridge with a cysteine residue naturally present in the active site of the protein, we optimised the active site by removing by directed mutagenesis a second cysteine residue (cys to ser mutation) which could interfere with the drug conjugation reaction. The drug is released in the target cell after endocytosis by the reducing conditions occuring in the lysosmes. On the other hand, as the optimal structures of the binding site are unknown, large combinatorial libraries of protein mutants were created and selected using the phage display technology which has become one of the most powerful methods of protein engineering to perform directed in vitro evolution of proteins. The filamentous bacteriophage M13 or fd is a virus infecting Gram negative bacteria. The genomic material of these bacteriophages consists of circular single stranded DNA enclosed into a proteinic coat composed, among others, of the products of genes III (g3p) and VIII (g8p). Active enzymes can be expressed on the bacteriophage surface in fusion with the coat protein pIII providing an interesting system in which the protein and its function are physically linked to the gene. Directed and random mutageneses generate large libraries of mutants from which clones of interest can be selected for binding to immobilised ligands or for new catalytic properties. The glutathione S-transferase P1-1 has been successfully displayed on the fd bacteriophage surface. Two libraries were generated by directed and random mutageneses inserting random peptides into an accessible loop of the glutathione S-transferase P1-1 (between residues N134 and Q135). In the first library (106 clones), the inserted peptides contain an RGD (Arg-Gly-Asp) motif found in most of the natural ligands of integrins receptors expressed on the surface of mammalian cells. The second library (109 clones) was created by insertion of a fully randomised heptapeptide X7 and selected on mammalian cells expressing the neurotensin receptor. Selections were performed on CHO-K1 cells to isolate clones displaying an optimal binding site allowing their recognition by an eucaryotic surface cell receptor. Selected RGD sequences were then cloned in GSTP1-1 gene (cys to ser mutant) which has been previously introduced into a pET expression system in order to produce large quantities of the carrier in E. coli. Purification of the carrier was achieved by affinity chromatography. The conjugation reaction was validated using a fluorescent molecule. The reaction showed to be specific to the cysteine residue and reversible in reducing conditions. Finally, the fluorescent carrier was tested on eucaryotic cells giving very interesting results.
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Boretti, M. (2003). Ciblage de molécules vers des cellules eucaryotes à l’aide d’un transporteur protéique construit par ingénierie de la glutathion S-transférase P1-1 humaine. https://hdl.handle.net/2078.5/97426