Methods and compositions for making vault particles in yeast
Disclosed herein are methods and compositions for making vault particles in yeast hosts and yeast vaults produced therefrom.
1. A yeast host cell, which comprises a nucleic acid sequence that encodes a major vault protein (MVP) sequence having 95-100% sequence identity to SEQ ID NO: 2, wherein said nucleic acid sequence comprises at least 95% sequence identity to SEQ ID NO: 1.
2. The yeast host cell according to claim 1 , wherein the yeast host cell contains multiple copies of the nucleic acid sequence.
3. The yeast host cell according to claim 1 , wherein the nucleic acid sequence is under the control of a promoter such as a constitutive promoter, an inducible promoter, or a yeast promoter.
4. The yeast host cell according to claim 1 , wherein the yeast host cell has been recombinantly modified to express one or more passenger peptides.
5. The yeast host cell according to claim 1 , wherein the yeast host cell is a microorganism belonging to the family Saccharomycetaceae.
6. A method of making a major vault protein (MVP) in a yeast host, which comprises culturing the yeast host cell according to claim 1 under conditions suitable for expression of the major vault protein.
7. A method of making a vault particle in a yeast host, which comprises culturing the yeast host cell according to claim 1 under conditions suitable for formation of the vault particle.
8. A method of producing a commercial-scale amount of vault particles, which comprises culturing the yeast host cell according to claim 1 in a cell culture medium to obtain a host cell culture that is at or near a stationary growth phase.
9. The method according to claim 7 , and further comprising extracting the vault particle from the yeast host cell or cells of the host cell culture.
10. The method according to claim 7 , and further comprising packaging one or more passenger molecules on or in the vault particle by (a) covalently linking the one or more passenger molecules to the N-terminus and/or C-terminus of the major vault protein, (b) mINT fusion packaging, and/or (c) passive packaging.
11. The method according to claim 7 , and further comprising packaging one or more mINT passenger molecules within the cavity of the vault particle.
12. A composition comprising one or more yeast host cells according to claim 1 , one or more major vault proteins made by culturing the one or more yeast host cells, and/or one or more vault particles expressed by the one or more yeast host cells.
13. The method according to claim 8 , and further comprising extracting the vault particle from the yeast host cell or cells of the host cell culture.
14. The method according to claim 8 , and further comprising packaging one or more passenger molecules on or in the vault particle by (a) covalently linking the one or more passenger molecules to the N-terminus and/or C-terminus of the major vault protein, (b) mINT fusion packaging, and/or (c) passive packaging.
15. The yeast host cell according to claim 1 , wherein the yeast host cell is a microorganism belonging to the genus Pichia.
16. The yeast host cell according to claim 1 , wherein the amino acid sequence of the major vault protein is SEQ ID NO: 2.
17. A yeast host cell comprising a nucleic acid sequence comprising SEQ ID NO: 1.
18. The yeast host cell according to claim 1 , wherein said nucleic acid comprises at least 96% sequence identity to SEQ ID NO: 1.
19. The yeast host cell according to claim 1 , wherein said nucleic acid comprises at least 97% sequence identity to SEQ ID NO: 1.
20. The yeast host cell according to claim 1 , wherein said nucleic acid comprises at least 98% sequence identity to SEQ ID NO: 1.
21. The yeast host cell according to claim 1 , wherein said nucleic acid comprises at least 99% sequence identity to SEQ ID NO: 1.