Progress 07/15/04 to 06/14/08
Outputs
OUTPUTS: Since this project did not work out as proposed, outputs achived are minimal. The major output achieved by this project is that it provided several students with research opportunities that they otherwise would not have have access to. Since no data was obtained, the results could not be disseminated. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major goals of this project were to overexpress and purify recombinant Mannheimia haemolytica MurA and use the purified protein in a high throughput approach to find novel inhibitors of this enzyme. While we were able to achieve some expression of the protein, levels achieved were far below the needed amounts for high throughput screening. Many different experimental approaches were investigated with no success realized. This was not anticipated by us as we and others have overexpressed MurA from other organisms with good success. The inability to obtain the needed amounts of MurA prevented us from progressing on this project. The only benefit to us from this project was that we were able to explore many different protein overexpression techniques and gained knowledge that will hopefully be applicable to other systems.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: We are currently in the second year of extension on this project. Our primary objective during this period has been to overexpress and purify enough recombinant MurA for high throughput screening of a chemical library to identify potential inhibitors. Unfortunately, we have been unsuccessful in achieving increased expression levels despite the different approaches examined. This setback is preventing us from moving forward on this project.
PARTICIPANTS: Dr. Scott Crupper, Professor, PI of this project. Various undergraduate student workers have participated in overexpression studies. These students were Microbiology majors generally at the junior-senior level.
PROJECT MODIFICATIONS: Since we have been unable to overexpress recombinant MurA to sufficient levels, we have completely revaluated expression conditions. To date, these changes have not proven beneficial. Furthermore, we have generated and are in progress of generating new genetic constructs that will be evaluated in the upcoming months.
Impacts
Findings from this report period have not had a positive effect on impacting the problem of shipping fever in cattle. If our expression problems cannot be overcome, we will not be able to discover a novel MurA inhibitor useful in the treatment of Mannheimia haemolytica infections in cattle.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
In this reporting period, our primary objective has been to overexpress murA in Escherichia coli and purify recombinant MurA for use in a high-throughput screen. Although we have successfully overexpressed murA, levels obtained are not sufficient for subsequent purification of recombinant MurA on the level needed for high-throughput ananlysis. While various experimental conditions for overexpression have been examined, none have been found acceptable.
Impacts
Shipping fever in cattle results in significant economic losses each year. The discovery of a novel MurA inhibitor could provide an alternative to current day therapeutics.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
The goal of Objective 1 is to overexpress and purify enzymatically active Mannheimia haemolytica MurA. To date, we have generated two different expression constructs and have evaluated their expression in a variety of different E. coli host strains. One construct expresses native MurA, while the other expresses MurA as a fusion with NusA. Furthermore, we have examined the co-expression of molecular chaperones in the same cell in order to increase expression. Our results to date indicate overexpression of each construct yields soluble MurA, but the construct in which MurA is expressed as a native protein (no fusion) results in better expression. Unfortunately, expression levels are not real high, and attempts to increase expression through manipulation of induction conditions have failed to yield positive results. Host strains other than E. coli BL21(DE3), as well as co-expression with different molecular chaperones, have no noticeable effect on expression levels. On a
small-scale, we have purified recombinant MurA and determined that it is enzymatically active. Attempts to scale up our procedures, however, have failed. In part, one reason for this lack of success has been equipment problems. These problems have recently been taken care of and now we are able to begin our purification on a larger scale again. The lack of significant overexpression is complicating our studies, but since we have demonstrated small-scale purification of active MurA, it is anticipated we will optimize our procedures to be successful on a larger scale. The goal of Objective 2 is to perform high-throughput screening of a chemical library in order to identify novel inhibitors of MurA. These studies have not been initiated as they are dependent on large quantities of purified MurA (Objective 1).
Impacts
If new inhibitors are found via the high-throughput screening, they could serve as a scaffold on which to design additional inhibitors of M. haemolytica MurA. Ultimately, this work could contribute to the development of a novel way to treat shipping fever in cattle.
Publications
- No publications reported this period
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