Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use
The present invention provides isolated polypeptides isolatable from a Staphylococcus spp. Also provided by the present invention are compostions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.
1. A method of isolating a metal-regulated polypeptide from Staphylococcus cells, the method comprising:
growing the Staphylococcus cells in a medium that comprises 2,2′-dipyridyl at a concentration of at least about 800 μM under conditions effective for the cell to express at least one metal-regulated polypeptide; and
isolating the at least one metal-regulated polypeptide from the cells.
2. The method of claim 1 wherein the medium comprises the 2,2′-dipyridyl at a concentration of about 1000 μM.
3. The method of claim 1 wherein the metal-regulated polypeptide is an exogenous polypeptide.
4. The method of claim 1 wherein the medium comprises the 2,2′-dipyridyl at a concentration of about 300 μg/mL.
5. A method of isolating a metal-regulated polypeptide from cells, the method comprising:
growing a seed stock of cells;
transferring at least a portion of the seed stock to a culture comprising a chelator at a concentration of at least about 300 μg/mL, thereby forming a working seed;
growing the working seed under conditions effective for the cells to express at least one metal regulated polypeptide; and
isolating the metal-regulated polypeptide from cells of the working seed.
6. The method of claim 5 wherein transferring at least a portion of the seed stock to a culture comprising a chelator at a concentration of at least about 300 μg/mL comprises serially subculturing cells in medium comprising increasing concentrations of chelator.
7. The method of claim 5 wherein transferring at least a portion of the seed stock to a culture comprising a chelator at a concentration of at least about 300 μg/mL comprises:
transferring at least a portion of the seed stock to a first subculture comprising a chelator at a concentration of at least about 300 μg/mL; and
transferring at least a portion of the first subculture to a second subculture comprising a chelator at a concentration greater than the concentration of chelator in the first subculture.
8. The method of claim 7 further comprising transferring at least a portion of the second subculture to a third subculture comprising a chelator at a concentration greater than the concentration of chelator in the second subculture.
9. The method of claim 8 wherein at least one subculture comprises a chelator at a concentration of at least 900 μg/mL.
10. The method of claim 8 wherein at least one subculture comprises a chelator at a concentration of at least 1200 μg/mL.
11. The method of claim 8 wherein at least one subculture comprises a chelator at a concentration of at least 1500 μg/mL.
12. The method of claim 8 wherein at least one subculture comprises a chelator at a concentration of at least 1800 μg/mL.
13. The method of claim 5 wherein the chelator comprises 2,2′-dipyridyl.
14. The method of claim 5 wherein the cells comprise gram positive microbes.
15. The method of claim 14 wherein the gram positive microbes comprise members of the family Micrococcaceae.
16. The method of claim 14 wherein the gram positive microbes comprise Staphylococcus species, Corynebacterium species, Mycobacterium species, or Erysipelothrix species.
17. The method of claim 16 wherein the gram positive microbe comprises Staphylococcus aureus.
18. The method of claim 5 wherein the metal-regulated polypeptide is an exogenous polypeptide.
19. A method of preparing antibody that specifically binds to a metal-regulated polypeptide natively expressed by Staphylococcus cells, the method comprising:
growing the Staphylococcus cells that express the metal-regulated polypeptide in a medium that comprises 2,2′-dipyridyl at a concentration of at least about 800 μM under conditions effective for the cell to express the metal-regulated polypeptide;
isolating the metal-regulated polypeptide from the cells; and
preparing antibody that specifically binds the isolated metal-regulated polypeptide.
20. The method of claim 19 wherein preparing antibody comprises preparing recombinant antibody.
21. The method of claim 19 wherein preparing antibody comprises:
administering an amount of the metal-regulated polypeptide effective to induce the animal to produce antibody that specifically binds to the metal-regulated polypeptide; and
isolating the antibody.
22. The method of claim 21 wherein the antibody is polyclonal antibody.
23. The method of claim 21 wherein the antibody is a monoclonal antibody.
24. The method of claim 19 wherein the metal-regulated polypeptide is an exogenous polypeptide.
25. The method of claim 19 wherein the medium comprises 2,2′-dipyridyl at a concentration of at least about 1000 μM.
26. The method of claim 19 wherein growing the cells comprises:
growing a seed stock of cells;
transferring at least a portion of the seed stock to a culture comprising 2,2′-dipyridyl at a concentration of at least about 300 μg/mL, thereby forming a working seed;
growing the working seed under conditions effective for the cell to express at least one metal regulated polypeptide; and
isolating the metal-regulated polypeptide from cells of the working seed.
27. The method of claim 26 wherein transferring at least a portion of the seed stock to a culture comprising 2,2′-dipyridyl at a concentration of more than about 300 μg/mL comprises serially subculturing cells in medium comprising increasing concentrations of “2,2′-dipyridyl”.
28. The method of claim 26 wherein transferring at least a portion of the seed stock to a culture comprising 2,2′-dipyridyl at a concentration of at least about 300 μg/mL comprises:
transferring at least a portion of the seed stock to a first subculture comprising 2,2′-dipyridyl at a concentration of at least about 300 μg/mL; and
transferring at least a portion of the first subculture to a second subculture comprising 2,2′-dipyridyl at a concentration greater than the concentration of 2,2′-dipyridyl in the first subculture.
29. The method of claim 28 further comprising transferring at least a portion of the second subculture to a third subculture comprising 2,2′-dipyridyl at a concentration greater than the concentration of 2,2′-dipyridyl in the second subculture.
30. The method of claim 29 wherein at least one subculture comprises 2,2′-dipyridyl at a concentration of at least 900 μg/mL.
31. The method of claim 29 wherein at least one subculture comprises 2,2′-dipyridyl at a concentration of at least 1200 μg/mL.
32. The method of claim 29 wherein at least one subculture comprises 2,2′-dipyridyl at a concentration of at least 1500 μg/mL.
33. The method of claim 29 wherein at least one subculture comprises 2,2′-dipyridyl at a concentration of at least 1800 μg/mL.