Characterization of Spermidine:spermine N1- acetyltransferase from the intestinal parasite Cryptosporidium parvum

Blog #2

November 26, 2012

Emmanuel Bujans

 

In my first blog post, I described the many issues as well as actions that need to be taken surrounding the organism Cryptosporidium parvum and the disease it causes, Cryptosporidiosis. Since the beginning of the semester, I have been working on cloning and expressing a new purified SSAT protein, however, the last protein I stored was found to be denatured.

Although I was at the final stages of purifying the SSAT protein, because of Hurricane Sandy all my samples were ruined. This occurred due to the power outage, which affected the -4 °C fridge where my temperature-dependent samples were being stored. Since there was no power, the heat in the fridge slowly rose to room temperature thus denaturing my samples. Denaturation of proteins usually occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between “side chains” including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. Therefore, a variety of reagents and conditions can cause denaturation. The common condition in this case was heat.

Even though horrible disasters may happen, which may impede one’s research, I have learned that I must continue past all those frustrations if I truly want to be successful. Therefore, for the next few weeks, I will restart the long process of cloning and expressing the SSAT protein. This is done by PCRing the desired known DNA sequence, then choosing one of the PCR samples containing the closest molecular weight size marker in a electrophoresis gel, DNA cleanup before ligation, ligation into a suitable vector, and transformation and screening to identify recombinant clones. This process usually takes about a full three weeks to complete.

Overall, as the semester has progressed, working with Dr. Nigel Yarlett has taught me how to apply knowledge learned in the classroom and relate it to microbial research. The next steps will be to perform experimental assays that measure the SSAT activity of the new purified protein in relation to C. parvum oocysts.

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