During this past semester, I mostly worked with the isolation of hsfi1 domains, particularly hsfi1 D10, with hopes of successfully isolating this one. Success of this purification would serve useful for future isolation of the D10-12 construct, which is known to be a more hydrophobic dimer. The problems we encounter throughout the isolation process ranged from domain solubilization to subsequent binding efficiency to the glutathione affinity column used, as well as optimal recovery of the pure domain. Unfortunately, our first attempts in obtaining soluble protein from our lysis procedure were not successful, primarily due to a poor protocol design. Because hsfi1 domains are highly insoluble, detergents are needed to aid in protein solubility. Sarcosyl is particularly used for this task. Unfortunately, Sarcosyl interferes with subsequent binding of the soluble protein once it is decanted to the affinity column. We were not aware of this at first. Triton X-100, a non-ionic detergent is used to precipitate Sarcosyl, forming large micelles that aid in Sarcosyl trapping, avoiding interference of Sarcosyl in the protein-binding step performed. In summary, the addition of proper amounts of Triton-X100 detergent after Sarcosyl treatment did indeed aid in protein solubility immensely.
Although we have been unable to optimize our GST-tagged hsfi1 D10 domain affinity to the glutathione sepharose column used, we have reason to believe a DTT treatment in our lysis procedure, prior to the Sarcosyl treatment described above might potentially solve this issue. Future experiments are needed to confirm this hypothesis.
Several enzymatic cleavage assays were performed in order to estimate optimal amounts of protease needed for complete cleavage of our GST-tagged construct. These experiments were successful and are described in detail in the final semester report of the project.
We noticed that once the protease cleavage treatment was performed and the cleaved mixture was once again eluted through the column for GST-tag / cleaved construct separation, the amounts of recovered construct from the column were not comparable to those observed prior to this final column separation step. We hypothesized that the construct could potentially have an affinity for the column matrix as well, even without its GST tag. This idea was suggested after acknowledging previous studies found in the scientific literature where hydrophobic constructs were found to bind the column matrix, as a consequence of inclusion bodies formed between them. Non-ionic detergent was used in some cases to aid in the solubility of the matrix/bound construct and subsequent elution. We performed this step in our own experiment and did, indeed, recover cleaved protein bound to the column matrix, something that was not previously thought possible for our constructs of interest in the lab until now.
In conclusion, optimization in the binding activity of the construct will be sufficient to write an official purification protocol for this domain. This one will be applied as well for the hsfiD10-12 construct.
Succesful isolation of this domain will allow us to use infrared correlational spectroscopy techniques (2D-COS) to study the binding interactions of this domain with centrin proteins. We plan on performing these biophysical studies during the course of next semester.
