Baculoviral expression and purification of dmsnapc for structural analysis

The small nuclear RNAs (snRNAs) are non protein coding RNA molecules that serve several essential roles such as pre-mRNA splicing, histone mRNA 3' end formation, rRNA processing and telomere maintenance. Most snRNAs are transcribed by RNA polymerase II, but other snRNAs are transcribed by RNA polymerase III. Although different polymerases are recruited for transcription, the promoter structures of all snRNAs are quite similar and lead to the recruitment of the same transcription factor, the small nuclear RNA activating protein complex (SNAPc). SNAPc is a multi-subunit complex that is essential to initiate transcription of small nuclear RNAs. It is highly conserved in all eukaryotes as far back as the ancient trypanosomes. SNAPc is composed of a core three subunits: SNAP190, SNAP50, and SNAP43. Research in our lab, utilizing the fruit fly model system, suggests that the differential recruitment of the RNA polymerases may be due to conformational differences in SNAPc when bound to the U1 and U6 proximal sequence elements (PSEs) in the Drosophila U1 and U6 promoters. Currently, no structural information exists for this essential protein to help understand this mechanism. It is the purpose of the work described in this thesis to express and purify Drosophila melanogaster SNAPc (DmSNAPc) for structural determination by X-ray crystallography. Protein crystallization requires a high yield of homogenously purified protein, which had not yet been accomplished in our lab for DmSNAPc. For the purpose of high level and simultaneous protein expression of the three DmSNAPc subunits within the same cells, a recently developed baculovirus system, the Multibac system, was implemented. This system required engineering and titering of a baculovirus that is capable of expressing large quantities of DmSNAPc when Spodoptera frugiperda moth (Sf9) cells were infected. Following the infection and protein expression, cells were lysed and DmSNAPc was subjected to several different chromatography methods for purification: anion exchange, nickel chelate, and size exclusion. Towards the end of the project, focus shifted to expression of one subunit, DmSNAP43, in order to optimize both protein expression and purification. Although expression and partial purification were achieved, production of suitable quantities of protein for crystallization purposes will require further optimization.