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Cutting a Cancer-Related Protein in Half and Re-ligating it to Identify its Role in Hormone-inducible Breast Cancer Open Access
Breast and ovarian cancers are known as one of the most aggressive types of cancers, either originating from DNA mutations, or hormone over-expression. Cancer involves the disruption of a cell-signaling pathway that affects cell life cycle, such as uncontrollable growth or loss of death. Hormone-inducible breast and ovarian cancers is associated with disruption of the interaction of the protein alpha4 with UBR5 and MID1. The C-terminal region of alpha4 interacts with the MID1 B-box1 domain and the UBR5 PABC domain. When PABC binds to alpha4, it disrupts the interaction between alpha4 and MID1 B-box1 domain. We hypothesize that this lost in alpha4-Bbox1 binding is due to conformational change of alpha4C caused by the binding of the PABC domain. To understand how the relay of conformational changes, we have engineered the C-terminal of alpha4 (105aa) as two separate, smaller proteins (alpha4CN, alpha4CC) that can later be stitched back to its full length domain using the Sortase A enzyme. To engineer alpha4 as two halves, the gene (DNA sequence) for each half of the protein was purchased from IDTDNA, then amplified using PCR. After PCR, the gene was cloned into a bacterial plasmid, which was then transformed into BL21DE3 E. Coli, a bacterial strain optimized for protein expression. The results show that we have successfully expressed and purified both alpha4CN and alpha4CC and are prepared to proceed with the Sortase A reaction.
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SyedA.pdf | 2019-05-16 | Open Access |
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