1-21. (canceled)
22. The UspA2 protein of claim 25 comprising less than about 0.1 endotoxin units per ug of UspA2 protein.
23. An isolated and purified gram negative bacteria protein obtained by a method for removing bacterial endotoxin from a gram negative bacterial protein solution, wherein the method uses immobilized metal ion affinity chromatography (IMAC) comprising the steps of:
(a) applying the Gram negative bacteria protein solution to an IMAC resin, wherein the resin is equilibrated in a buffer;
(b) eluting the endotoxin with the buffer of step (a), wherein the protein remains bound to the resin;
(c) eluting the protein in an elution buffer comprising glycine; and
(d) collecting the eluted protein,
wherein the concentration of the endotoxin in the protein solution is effectively reduced by a factor of at least 200 and protein recovery is greater than at least 60%.
24. The protein of claim 24, wherein the Gram negative bacteria is Moraxella catarrhalis.
25. The protein of claim 25, wherein the protein is UspA2.
27. The protein of claim 23, wherein the glycine of step (c) is at a concentration of at least 1 mM to at least about 500 mM.
28. The protein of claim 23, wherein the buffer of step (a) is selected from the group consisting of sodium phosphate, sodium acetate, 1,4-piperazinebis-(ethanesulfonic acid) (PIPES), N-(2-acetamido)imino-diacetic acid (ADA) and N-(2acetamido)-2-aminoethanesulfonic acid (ACES).
29. The protein of claim 23, wherein the buffer of step (a) further comprises NaCl at a concentration of at least about 100 mM to about 500 mM.
30. The protein of claim 23, wherein the elution buffer of step (c) is selected from the group consisting of sodium phosphate, sodium acetate, imidazole, histidine, ammonium chloride, PIPES, ADA and ACES.
31. The protein of claim 23, wherein the elution buffer of step (c) further comprises NaCl at a concentration of at least about 100 mM to about 500 mM.
32. The protein of claim 28, wherein the buffer of step (a) is sodium phosphate at a concentration of at least about 1 mM to about 50 mM and has a pH of about 6.5 to about 7.8, the buffer further comprising about 150 mM NaCl and 0.1% Triton-X.
33. The protein of claim 30, wherein the elution buffer of step (c) is sodium phosphate at a concentration of at least about 1 mM to about 50 mM and has a pH of about 6.5 to about 7.8, the buffer further comprising about 150 mM NaCl, 0.04% Triton-X and 100 mM glycine.
34. The protein of claim 32, wherein the sodium phosphate has a concentration of about 10 mM and a pH of 6.8.
35. The protein of claim 33, wherein the sodium phosphate has a concentration of about 10 mM and a pH of 6.8.
36. The protein of claim 23, wherein the resin is charged with Cu2+, Ni2+, Co2+, Fe2+, Mn2+, Ti2+, Cd2+, Mg2+, Zn2+ or Fe3+.
37. The protein of claim 36, wherein the resin is charged with 20 mM cupric sulfate.
38. The protein of claim 23, wherein the buffer of step (a) and the elution buffer of step (c) each further comprise a mobile phase modifier selected from the group consisting of urea, isopropanol, ethanol, methanol, ethylene glycol and a detergent.
39. The protein of claim 23, wherein the concentration of the endotoxin in the protein solution is effectively reduced by a factor of at least 2,500 and protein recovery is greater than at least 90%.
40. The protein of claim 23, wherein the resin is washed prior to step (a) with at least 3 to 10 resin volumes of the buffer used in step (c) and then washed with 1 to 10 resin volumes of the buffer used in step (a), wherein the washes remove excess copper from the resin.
41. The protein of claim 23, wherein the protein solution is diluted in a buffer comprising 10 mM sodium phosphate, 150 mM NaCl and 0.25% Triton-X at pH 6.9, before proceeding to step (a).
42. The protein of claim 23, wherein the protein is further purified by ultrafiltration or filtration.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A device for capturing a biological material from a fluid specimen and lysing said fluid specimen at rapid flow rate and low pressure, the device comprising:
a first chamber containing:
a portion of said fluid specimen;
a solid phase material that has an affinity for the biological material in said fluid specimen;
a first fitting attached to an inlet opening of the first chamber, the first fitting having a first thin filter insert that retains the solid phase material while allowing rapid flow of the fluid specimen at low pressure; and
a second fitting attached to an outlet opening of the first chamber, the second fitting having a second thin filter insert that retains the solid phase material while allowing rapid flow of the fluid specimen at low pressure; and
a second chamber fluidly connected to said first chamber, said second chamber comprising:
a portion of said fluid specimen;
a lysing medium comprising a particulate material; and
an impeller, wherein said impeller is configured to impart rotational motion to said lysing medium to mechanically lyse said fluid specimen.
2. The device of claim 1 wherein said second chamber is connected to said inlet opening of said first chamber.
3. The device of claim 1 wherein the solid phase material has an affinity for at least one selected from the group consisting of a nucleic acid, a protein, a polypeptide, a His-tagged protein, a His-tagged polypeptide, a GST-tagged protein, a GST-tagged polypeptide, streptavidin, biotin, Calmodulin, an antigen, an antibody, an immunoglobulin, albumin, double stranded DNA, an RNADNA heteroduplex, maltose binding protein, FLAG-tagged protein, FLAG-tagged polypeptide, MYC-tagged protein, MYC-tagged polypeptide MYC, a glycoprotein, a SNAP-tag, an enzyme, a lipid-containing biological material, a glycosylated protein, a phosphorylated protein, and a microorganism having a high cell wall lipid content.
4. The device of claim 1 wherein the solid phase material has an affinity for more than one biological material.
5. The device of claim 1 wherein the solid phase material is a particulate or a bead.
6. The device of claim 5 wherein the particulate or bead has a diameter or lateral dimension of at least 10 \u03bcm.
7. The device of claim 1 wherein the first and second filter inserts have a pore size between about 10 \u03bcm and about 200 \u03bcm.
8. The device of claim 1 wherein the filter inserts are wire mesh inserts.
9. The device of claim 8 wherein the wire mesh inserts are stainless steel.
10. The device of claim 1 wherein the filter inserts are plastic mesh.
11. The device of claim 1 wherein said second chamber is connected to said outlet opening of said first chamber.
12. A method of capturing a biological material from a fluid specimen and lysing said fluid specimen at rapid flow rate and low pressure, the method comprising:
introducing the fluid specimen containing the biological material into a first chamber of a device, the first chamber containing a solid phase material that has an affinity for the biological material, the device having a first fitting attached to an inlet opening in the first chamber and a second fitting attached to an outlet opening in the first chamber, the first fitting and the second fitting having filter inserts that retain the solid phase material while allowing rapid flow of the fluid specimen through the filter insert; and
flowing the fluid specimen containing the biological material through the solid phase material at low pressure and rapid rate, the fluid specimen exiting the device via the outlet opening;
said method further comprising:
lysing the fluid specimen, prior to introducing the fluid specimen into the first chamber, in a second chamber fluidly connected to said inlet opening of said first chamber, said second chamber comprising:
a lysing medium comprising a particulate material; and
an impeller, wherein said impeller is configured to impart rotational motion to said lysing medium to mechanically lyse said fluid specimen.
13. The method of claim 12 wherein lysing the fluid specimen comprises agitating the specimen in the second chamber with a medium that includes a particulate material to mechanically lyse the specimen.
14. The method of claim 12 wherein introducing a fluid specimen into the first chamber of a device includes introducing the specimen into a first chamber containing a solid phase material having an affinity for at least one selected from the group consisting of a nucleic acid, a protein, a polypeptide, a His-tagged protein, a His-tagged polypeptide, a GST-tagged protein, a GST-tagged polypeptide, streptavidin, biotin, Calmodulin, an antigen, an antibody, an immunoglobulin, albumin, double stranded DNA, an RNADNA heteroduplex, maltose binding protein, FLAG-tagged protein, FLAG-tagged polypeptide, MYC-tagged protein, MYC-tagged polypeptide MYC, a glycoprotein, a SNAP-tag, an enzyme, a lipid-containing biological material, a glycosylated protein, a phosphorylated protein, and a microorganism having a high cell wall lipid content.
15. The method of claim 12 wherein introducing a fluid specimen into the first chamber of a device includes introducing the specimen into a first chamber containing a solid phase material having an affinity for more than one biological material.
16. The method of claim 12 wherein introducing a fluid specimen into the first chamber of a device includes introducing the specimen into a first chamber containing a solid phase material comprising a particulate or a bead.
17. The method of claim 16 wherein introducing a fluid specimen into the first chamber of a device includes introducing the specimen into a first chamber containing a solid phase material comprising a particulate or bead having a diameter or lateral dimension of at least 10 \u03bcm.
18. The method of claim 12 wherein introducing a fluid specimen into the first chamber of a device having a fitting having a filter insert includes introducing the specimen into a device having a filter insert of pore size between about 10 \u03bcm and about 200 \u03bcm.
19. The method of claim 12 wherein introducing a fluid specimen into the first chamber of a device having a first fitting and a second fitting having filter inserts includes introducing the specimen into a device having a wire mesh insert.
20. The method of claim 19 wherein introducing a fluid specimen into the first chamber of a device having a first fitting and a second fitting having filter inserts includes introducing the specimen into a device having a stainless steel mesh insert.
21. The method of claim 12 wherein introducing a fluid specimen into the first chamber of a device having a first fitting and a second fitting having filter inserts includes introducing the specimen into a device having a plastic mesh insert.
22. The method of claim 12, further comprising:
eluting the biological material captured on the solid phase material by flowing an elution medium through the solid phase at low pressure and rapid rate; and
collecting an effluent containing the biological material, the effluent exiting the device via the outlet opening.