Optimization of Buffering Capacity to Increase Protein Expression

In order to perform structural studies on proteins using NMR, proteins must be recombinantly expressed in isotopically enriched minimal media. One of the problems researchers face in this process is protein yield. For NMR studies, milligram quantities of protein are required. Using an Escherichia coli expression platform offers many benefits, including high yield. However, as the cell density increases and the metabolic cellular machinery shift to protein over-expression, the pH of the media decreases, leading to environmental stress (?) for the E. coli. Our hypothesis is that doubling the buffering capacity of the minimal defined (M9) media will increase yield of recombinantly expressed protein in E. coli. To test this hypothesis, we have expressed one protein, known as fortilin, in M9 minimal media. We monitored how the concentration of buffering salts in the M9 minimal media affects the pH stability of the cell culture during growth phase and after expression is induced. We are looking at how pH stability of the minimal media relates to protein yield. Our future work includes expressing protein in 2X M9 mineral media, where the salt concentrations have been doubled. The media that has a higher buffering capacity should be more resistant to pH change, and produce a greater yield of protein