Wong Jian, Yuan
(2008)
Over-expression of human y-box binding proteini:
a comparison between mammalian and bacterial expression system.
Other.
Universiti Sains Malaysia.
(Submitted)
Abstract
The human Y -box protein I (YB- I) is a class of proteins involved in transcriptional
and translational regulation of a wide range of genes. YB-1 is implicated in regulation of
multiple cellular functions. Previous studies found that the YB-1 expression is upregulated
in many tumours and is highly correlated with adverse cancer prognosis and resistance to
cancer drugs. In order to further explore and understand the role of YB-1 in terms of
transcriptional and translational regulation in development of malignancy tumours, an overexpression
assay has been carried out. Two different recombinants YB- I plasmid, pGEX-
2TK-YB-1 and pREP-YB-1 were used to carry out over-expression assay in bacterial
(Escherichia coli, BL-21) and mammalian system (He La cancer cells) respectively. The
comparison was analyzed by western blot analysis using a YB-1 polyclonal antibody. The
over-expression results show a different migration of YB-1 protein in bacterial and
mammalian expression system compared to their actual theoretical size. Meanwhile, overexpression
assay involved transfection of pREP-YB-1 into He La cells using calcium
phosphate transfection method. Due to poor reproducibility and rare quantitative studies on
the mechanisms of the calcium phosphate transfection, trouble shooting was done and the
optimization steps were suggested in the discussion of this research.
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