Protein interactome mapping of GTPBP9 (OLA1) to determine role in ribosome biogenesis
MetadataShow full item record
GTPBP9, originally only a predicted protein, is a poorly understood protein. GTPBP9 has only recently begun to be more specifically classified via its close relation to other better understood proteins, namely those of the Obg-like ATPase family. The yeast homologue of GTPBP9 has been implicated in ribosome biogenesis; as such, we were interested in the study of its function in metazoan cells. Traditionally, protein function is determined through analysis of its folding pattern, primary amino acid sequence and other characteristics unique to the protein. We will instead determine the probable function of our target protein GTPBP9 (OLA1) through protein interactome mapping. Rather than study a protein in isolation, the process of interactome mapping identifies protein activity by analyzing in vivo protein complexes within which a protein functions. Proteins of similar activity cluster together to create these complexes that provide greater functional efficiency. Classifying the interacting proteins in a complex helps with our understanding of our target protein’s probable activity. To identify and purify our target protein in complex, we utilized several novel techniques available for targeted gene manipulation. CLEP tagging is the insertion of an epitope tag, or an antibody target region, via targeted sequence insertion upstream of a gene’s stop codon. CLEP tagging differs from previous epitope tagging techniques in that it minimally alters the native gene sequence by inserting the tagging sequence just prior to the stop codon rather than replacing the entire gene sequence with one intronless homologue. Our lab has previously demonstrated success with insertion of TAP-tag sequences for protein identification and isolation. Thus, we chose to use a TAP-tag construct containing neomycin resistance as our tagging construct. Utilizing the bacterial Red DNA repair system enzymes, we inserted our TAP-tag construct into a BAC vector host via a process called recombineering. Recombineering utilizes the Red DNA repair system to initiate homologous recombination between transgenes and bacterial chromosomes. The use of recombineering allowed us to insert our TAP-tag construct into the Gallus gallus GTPBP9 sequence hosted in a BAC vector. Electroporation of chicken DT40 pre-B cells in the presence of these altered BAC vectors triggered a second round of homologous recombination, this time between the Gg regions of the BAC vectors and the DT40 cell chromosome. This resulted in DT40 cells expressing TAP-tagged GTPBP9, confirmed via Western Blot analysis. The expression of the TAP-tag on our target protein allowed for the easy isolation of our protein in complex from cell culture via binding affinity and column chromatography. Fractionation analysis and mass spectrometry analyses of both GTPBP9 and the proteins isolated in complex with it would allow for greater understanding of GTPBP9’s probable role in cellular functioning. However, by the time we were able to perform such analyses on our samples, the cells had undergone gene silencing; thus our construct was unable to be isolated. Because of our success in inserting the construct into metazoan cell lines and the nature of our time constraints, we hope that future attempts will experience greater success in maintaining TAP-tag expression.