As far as the progress of my project, based on Augusts’ objectives, I believe it is reflected somewhere between 25-30%. I recently worked on a large scale purification of hsfi1 D(10-12). Although we had previous knowledge that this construct is fairly insoluble, we were not at all prepared for the intricacies involved in isolating it. Kilmartin et.al have isolated the construct previously and, in fact, published their own set of results using the same construct we are currently trying to purify. This group points out that a modified version of Frangioni and Neel’s GST-tagged protocol was used to increase the solubility of the construct, as well as its affinity and consequent isolation. We were able to perform a successful isolation of hsfi1 D(10) a few months ago.
Although the isolation of a single hsfi1 domain was a big accomplishment for us, it is critical that we purify both domains at once, in order to gain a deeper understanding of the dynamics involved in hsfi1-hcentrin binding. Nonetheless, even a single added domain, as we noted, can increase the insolubility of the construct dramatically, making it extremely difficult to purify and isolate. Even various consecutive lysis procedures in the protein pellet have not shown promising results. This past week we expressed the single hsfi1 D(10) construct, which we plan on purifying in large yields, in order to gain some insight into how this construct behaves, and move on the hsfi1 D(10-12) construct.
We have also proposed using alternative methods for purifying these constructs, such as the use of a denaturing agent to increase the solubility of our-now- denatured protein, with hopes of renaturing the construct again once isolated. Nonetheless, not all constructs successfully fold back to their native state once denatured under certain conditions. A re-evaluation of the amino acid sequence of both constructs might prove beneficial, as sometimes adding or removing certain amino acids from the sequence being expressed can increase the construct solubility.
In other news, I am still learning how to perform spectroscopic studies using 2D-correlational spectroscopy. I will perform a couple of dynamic studies using various hcentrin constructs we’ve isolated. Hopefully, once the hsfi1 construct has been isolated, we will then perform the same 2D-COS studies with the hsfi1-hcentrin complexes. That is really the goal of this big project. Understanding the dynamics of these proteins allow further understanding of those events involved during cell-division events.
