Pre-mRNA splicing is a regulated process that removes introns and brings together the exons of transcribed DNA to create mRNA that can then produce protein.[1] The goal of this research project was to elucidate the role of PRCC (papillary renal cell carcinoma) protein in pre-mRNA splicing in order to further understand biological regulation of splice choice. This involved determining the relationship between PRCC and another cyclophilin, PPIL2 (peptidyl-prolyl isomerase-like 2), which has been shown to interact with PRCC and has proven influential in the regulation of splice choice in in vitro assays.   The SURF 2013 research project was focused on generating purified samples of human PRCC for use in crystallization, splicing assays, and protein:protein interaction studies. Generally, the Davis Lab generates highly pure protein via a five-step �pipeline� consisting of: 1) Construct Design, 2) Ligation-independent Cloning and Colony PCR, 3) TestEx (Small-scale Protein Expression), 4) LEX (Large-scale Protein Expression), and 5) Protein Purification. Previously, studies carried out by the Davis Lab indicated that that two PRCC constructs, PRCC 1-172 and 1-204, could be prepared in the presence of a GST vector, but not without this stabilizing protein. I successfully replicated these results using large-scale expression and purification protocols. However, when GST was removed from the large-scale samples of PRCC to further the purification, PRCC's concentration decreased dramatically. This was attributed to PRCC's instability, which may have led to protein degradation. In the future, PRCC will be co-expressed with PPIL2 in an attempt to stabilize selected constructs of PRCC.[2][1] Wikipedia. http://en.wikipedia.org/wiki/RNA_splicing (accessed June-August 2013).[2] I would like to acknowledge the contribution of my mentor, Dr. Tara Davis, in composing this abstract.

Characterization of proteins that regulate pre-mRNA splicing

247th National Meeting (2014)

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