|dc.description.abstract||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.||en