KSEF R&D Excellence: Improved Recovery of Engineered Pharmaceutical Proteins from Tobacco Plant Extract

Grants and Contracts Details


Ob1ectives The objective of this project is to test the hypothesis that engineered proteins containing a histidine tag can be purified from tobacco plant extract using the economically attractive method of foam fractionation. The specific aims are to: 1. Add a histidine-tagged protein to increasingly complex solutions, (a) a buffered solution, (b) E. coli extract, and (c) tobacco plant extract, and then isolate the histidine-tagged protein from each of these solutions by foam fractionation, employing a surfactant that will bind the protein through a Ni2+chelating bridge; () 0 Crofcheck et al. -6 2. Determine if the protein retains its structure and function following the foam fractionation separation process; 3. Adjust the solution and column parameters to optimize the recovery of the histidinetagged protein; and 4. Develop a model to aid in further analysis and scale-up. The overall objective will be addressed with the following specific objectives: determine the foam fractionation conditions under which the enrichment, recovery, and retention of protein activity of histidine-tagged proteins in the presence of the DCL surfactant are maximal, from the following protein systems: (a) a buffered solution, (b) the extract obtained from an E. coli culture, and (c) the extract obtain from tobacco plants. In addition, studies will be performed without the addition of DCL to investigate the foaming ability of just the histidine-tagged protein. If the histidine-tagged protein does not foam well, then a pre-foaming step before the addition of DCL may be used to remove other surface-active proteins that might otherwise interfere with the foaming. The recovery of a histidine-tagged protein using a foam fractionation has not been investigated and the removal of a specific protein from plant extract can be very complex. Therefore, the fIrst system under investigation will be a simple buffered solution. From this test, the general operating conditions will be verified and the maximum foam fractionation performance will be established, since the performance is expected to decrease as the complexity of the protein mixture increases. Recovering the tagged protein from the E. coli culture will mimic the removal of the protein as if the bacteria had produced it. In this way, the foaming performance can be established with respect to a biological system with well-established pre-foaming conditioning steps (i.e., more complex than the buffered solution, but not as complex as the plant extract system). Finally, the histidine-tagged protein will be added to and recovered from tobacco plant extract. The pre-foaming conditioning steps are less established for the plant extract, and may need to be optimized along with the foaming conditions.
Effective start/end date7/1/029/30/04


  • KY Science and Technology Co Inc: $50,393.00


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.