Methods for making compositions including gram negative microbial polypeptides
AssigneeEpitopix, LLC
InventorsDaryll A. Emery, Darren E. Straub, Donavan E. Zammert, Gayla K. Kallevig
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Abstract
The present invention provides methods for making compositions including siderophore receptor polypeptides and porins from gram negative microbes. The methods include providing a gram negative microbe, disrupting the microbe, solubilizing the disrupted microbe, and isolating the polypeptides.
Claims86
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe in a buffer, wherein the gram negative microbe is present in the buffer at a concentration of between about 720 grams of microbe per 1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000 milliliters of buffer;
- solubilizing the disrupted gram negative microbe for greater than about 24 hours in a solution comprising sarcosine, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe;
- and
- isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe;
- solubilizing the disrupted gram negative microbe in a solution comprising sarcosine, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe;
- and
- isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe;
- solubilizing the disrupted gram negative microbe for greater than about 24 hours;
- and
- isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe in a buffer, wherein the gram negative microbe is present in the buffer at a concentration of between about 720 grams of microbe per 1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000 milliliters of buffer;
- solubilizing the disrupted gram negative microbe;
- and
- isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe;
- solubilizing the disrupted gram negative microbe for greater than about 24 hours in a solution comprising sarcosine;
- and
- isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe in a buffer, wherein the gram negative microbe is present in the buffer at a concentration of between about 720 grams of microbe per 1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000 milliliters of buffer;
- solubilizing the disrupted gram negative microbe for greater than about 24 hours, wherein a ratio of the sarcosine to gram weight of disrupted gram negative microbe is between about 0.8 gram sarcosine per about 4.5 grams of disrupted gram negative microbe and about 1.2 grams sarcosine per about 4.5 grams of disrupted gram negative microbe;
- and
- isolating molecules of the gram negative microbe, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides, at least two porins and lipopolysaccarhide at a concentration of no greater than about 10.0 EU/ml.
A method for isolating outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins, the method comprising
- providing a gram negative microbe;
- disrupting the gram negative microbe;
- solubilizing the disrupted gram negative microbe in a detergent for greater than about 24 hours;
- and
- isolating molecules of the gram negative microbe by centrifugation, wherein the isolated molecules comprise outer membrane polypeptides comprising at least two siderophore receptor polypeptides and at least two porins.
Dependencies are inferred from the claim text.