GW Work

 

Cutting a Cancer-Related Protein in Half and Re-ligating it to Identify its Role in Hormone-inducible Breast Cancer Open Access

Downloadable Content

Download PDF

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.

Author Language Keyword Type of Work Rights statement GW Unit Persistent URL
License

Relationships

Items