Method for the preparation of virus-like particles (VLPS) comprising heterologous multiple membrane spanning proteins
View Patent ↗Enveloped virus vectors are described which comprise a cellular virus receptor protein and which are capable of fusing with a cell which comprises a viral envelope protein to which the cellular virus receptor protein is cognate. Enveloped virus vectors comprising a plurality of cellular virus receptor proteins are also described. Methods for making the enveloped virus vectors are described, as are methods of using the enveloped virus vectors. The invention further relates to a lipoparticle comprising a membrane spanning protein, and the lipoparticle can be attached to a sensor surface. The invention relates to methods of producing and using the lipoparticle to, inter alia, assess protein binding interactions.
1. A method of producing a virus-like particle comprising an enveloped virus core and a heterologous multiple membrane spanning protein comprising:
contacting a cell that expresses an enveloped virus core with a nucleic acid sequence encoding the heterologous multiple membrane spanning protein under conditions sufficient to express the multiple membrane spanning protein in the cell
culturing the cell expressing the enveloped virus core and the heterologous multiple membrane spanning protein in media under conditions sufficient to produce the virus-like particle comprising the enveloped virus core and the heterologous multiple membrane spanning protein; and
isolating the virus-like particle comprising the enveloped virus core and the heterologous multiple membrane spanning protein.
2. The method of claim 1 , wherein the cell transiently expresses the enveloped virus core.
3. The method of claim 1 , wherein the cell stably expresses the enveloped virus core.
4. The method of claim 1 , wherein the cell is contacted with a nucleic acid sequence encoding the enveloped virus core prior to the cell being contacted with the nucleic acid sequence encoding the multiple membrane spanning protein.
5. The method of claim 4 , wherein the contacting the nucleic acid sequence encoding the enveloped virus core with the cell comprises transfecting the cell with the nucleic acid sequence encoding the enveloped virus core.
6. The method of claim 1 , wherein the nucleic acid sequence encoding the heterologous multiple membrane spanning protein is DNA.
7. The method of claim 1 , wherein the nucleic acid sequence encoding the heterologous multiple membrane spanning protein is a vector.
8. The method of claim 1 , wherein the nucleic acid sequence encoding the heterologous multiple membrane spanning protein is a plasmid.
9. The method of claim 1 , wherein the contacting the cell with a nucleic acid sequence encoding the heterologous multiple membrane spanning protein comprises transfecting the cell with the nucleic acid sequence encoding the heterologous multiple membrane spanning protein.
10. The method of claim 1 , wherein the cell is a HEK-293 cell.
11. The method of claim 1 , wherein the isolating comprises isolating the media comprising the particles.
12. The method of claim 1 , wherein the isolating comprises isolating the media comprising the particles and centrifuging the media comprising the particles to pellet said particles.
13. The method of claim 12 , wherein the pelleted particles are suspended in phosphate buffered saline (PBS).
14. The method of claim 1 , wherein the multiple membrane spanning protein is selected from the group consisting of a G-protein coupled receptor, a transporter protein, and an ion channel protein.
15. The method of claim 1 , wherein the multiple membrane spanning protein is a G-protein coupled receptor.
16. The method of claim 1 , wherein the enveloped virus core is an enveloped virus core selected from the group consisting of a murine leukemia virus enveloped virus core, a human immunodeficiency virus enveloped virus core, a rabies virus enveloped virus core, a Rous sarcoma virus enveloped virus core, and a vesicular stomatitis virus enveloped virus core.
17. The method of claim 1 , wherein the multiple membrane spanning protein is selected from the group consisting of CCR5, CXCR4, MCAT-1, CXCR2, CXCR3, mu-opoid receptor, and KCNH2 potassium channel protein.