| US 7,553,634 B1 | ||
| Extraction of integral membrane proteins | ||
| Sanjay Lakhotia, Danville, Calif. (US); and Michael R. Biehl, Sanford, N.C. (US) | ||
| Assigned to Wyeth Holdings Corporation, Madison, N.J. (US) | ||
| Appl. No. 10/19,163 PCT Filed Jun. 20, 2000, PCT No. PCT/US00/17019 § 371(c)(1), (2), (4) Date Dec. 20, 2001, PCT Pub. No. WO01/00668, PCT Pub. Date Jan. 04, 2001. |
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| Int. Cl. C12P 1/00 (2006.01); A61K 39/00 (2006.01); A61K 39/21 (2006.01) | ||
| U.S. Cl. 435—41 [424/184.1; 424/208.1] | 16 Claims |
| 1. A process for extracting native or recombinantly-expressed, gram-negative inner membrane proteins from bacteria or bacterial
host cells containing a recombinant vector by differential detergent tangential flow diafiltration, which comprises:
(a) lysing bacteria or bacterial host cells containing a recombinant vector in a fermentation broth;
(b) diafiltering the lysed fermentation broth from (a) with a buffer which is not retained by the diafiltration membrane,
wherein said buffer removes intracellular and extracellular contaminants through the permeate, and using a chelating agent
to prevent proteolysis;
(c) diafiltering the lysate from (b) with a detergent and a buffer which is not retained by the diafiltration membrane, wherein
said detergent solubilizes and removes inner membrane proteins, and using a divalent cation to stabilize the outer membrane
proteins, thereby preventing their solubilization; and
(d) collecting the inner membrane proteins removed in (c).
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