Progress Report 1 – Sravani Mannuru
10/18/11
R0, N58, Renalase peptide

Progress, Results and Discussions
Biotinylation of Renalase Peptide
The renalase peptide was biotinylated to produce a stable product. The ester group of the biotinylation reagent reacts with the amino acid lysine from the protein and attacks primary amines. The biotinylated peptide was purified to remove any remaining biotin that was not bounded to the protein. It was assumed that 90% of the biotinylated peptide was recovered (look at Problems Encountered section). The biotinylated peptide was aliquoted into manageable 400 pmol proportions to avoid excessive freeze and thaw cycles.


Problems Encountered
Since working with a peptide, the full-length renalase amino acid sequence is not available to obtain the real concentration. This means the renalase peptide does not contain the amino acids needed for spectroscopy to absorb at 280. However, the company which supplied the peptide indicated the concentration to be 1 mg/ml. On good faith, this concentration was used to biotinylate the peptide. One of the last steps of biotinylation is to figure how much of the peptide/protein is recovered. Since I cannot again spec the solution, I assumed that 90% of the peptide was recovered- meaning 3600 pmol remained from a starting of 4000 pmol that was biotinylated.

Conclusion and Future Work
The renalase peptide was biotinylated which yielded a 90% recovery. This was aliquoted into 9 - 400 pmol tubes and stored in the -20*C fridge. Future work includes starting the initial round (R0) using PBS as the selection buffer and N58 as the RNA pool. If I can make at least 3 rounds of selection work, I may switch over to perform selection on the full-length renalase protein (since most aptamers selected for peptides have been unsuccessful). As a test run we will see how the peptide works before pursuing a full-length renalase protein.

“Bonus” Work
I have mentored other aptamers students’ by showing them how to set up and run a PAGE. Also, I have made 3.8% acrylamide gel for the lab to use.

Here is a link to my abstract.