At present mammalian cell factories are being employed for recombinant protein production. However, the yields of proteins produced from such systems are often poor. This thesis describes experiments to study the effects of altering targeting signals (signal peptide) and 3’ untranslated regions (3’UTR) in an expression vector on protein expression. A variety of gene constructs containing Gaussia princeps luciferase as reporter were created using a seamless cloning method. In these constructs a variety of signal peptides, some with altered hydrophobicity, were combined with the Gaussia luciferase coding region and either the native Gaussia luciferase or human albumin 3’UTR. These were then transfected into CHO AA8 Tet-Off cells to measure how modification of the signal peptide/3’UTR affects protein expression. The results indicate that the Albumin 3’UTR, in conjunction with an appropriate signal peptide, boosts protein production by approximately 3 fold compared to the native Gaussia luciferase 3’UTR. Deletion analysis of the Albumin 3’UTR showed that deletion of regions 1-50, 1-100, 1-150, 101-150 significantly reduces protein production compared with deletion of regions 51-100, 51-150 and 1-50&101-150. Interestingly, mRNA abundance levels were significantly decreased for constructs containing deletions in regions 1-50, 1-150 and 1-50&101-150. UV Cross linking and electrophoretic mobility gel shift competition assays showed strong competition by RNA transcripts from the deletion construct 1-50, which was then used as bait for isolating bound protein/s from a CHO cell extract. Three proteins, including CUG-BP1 an RNA-binding protein involved in mRNA stability and translation were identified by mass spectrophotometry analysis. Knock down of CUG-BP1 expression using siRNA, led to impairment of complex formation between CHO cell protein extract and Albumin 3’UTR RNA transcripts, and in addition it led to an increase in the reporter activity and mRNA expression level in cells expressing the reporter gene with the full length Albumin 3’UTR and deletion variant 51-100. It is hypothesised that the differences in mRNA expression levels and secreted luciferase activity were due to CUG-BP1 binding to the Albumin 3’UTR. Further work is needed to explore the effects of CUG-BP1 on mRNA translation and stability.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594496 |
| Date | January 2011 |
| Creators | Khazaipoul, Siavash |
| Publisher | University of Newcastle upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10443/2181 |
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