Optimization of Metacaspase Protein Purification

As members of the caspase (cysteine-dependent aspartate-directed proteases) family, metacaspase proteins are active in proteolysis, helping to cleave proteins in cells to regulate programmed cell death, cell proliferation, and  relieve endoplasmic reticulum (ER) stress. The use of various purification methods allows a desired protein to be isolated from a mixture of protein and non-protein components. The metacaspase of interest in my work is ScMCA-Ia from the fungus Schizophyllum commune. Currently, we purify ScMCA-Ia using nickel affinity chromatography since the metacaspase has a His-tag, allowing it to bind to the nickel beads. This purification method is most effective when the protein being targeted has a specific affinity that is unique from the other proteins in the mixture so as not to harvest contaminants. My project aims to develop a second purification step to increase the purity of the samples produced via nickel affinity chromatography that could then be scaled up to yield a greater amount of purified protein. I am using ChromLab software to program fast protein liquid chromatography (FPLC) methods, and varying experimental conditions such as buffer pH, EDTA presence, the elution salt gradient and injected protein concentration to identify the optimal environment. Once the eluted samples are of the desired purity, the procedure can be scaled up to increase the yield. With a large enough yield, we can use x-ray crystallography to determine the structure of ScMCA-Ia. From my available data, optimal conditions were determined to be highly concentrated FPLC protein injections, with buffers adjusted to pH 8.0 without EDTA. It has also been shown that the samples eluted from the FPLC are of higher purity than those that have only been purified using nickel affinity chromatography. Currently, work is being done to replicate the data and verify these results. In addition to adjusting the pH of the buffers, EDTA has been added as it has since proven to prevent self cleavage of the metacaspase prior to being loaded onto the column. In the FPLC ChromLab software, I am adjusting the salt gradient during elution in an attempt to further separate the metacaspase from contaminants that may be eluted under similar salt concentrations as it.