Part:BBa_K2520008
CMV-Tri display-hGH
This device is a complete system for displaying proteins on cells' membrane. We combined three independent systems into one, which allows for the expression of three unique proteins. The Igk leader (BBa_K2520024,
BBa_K2520028, BBa_K2520029) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (BBa_K2520026, BBa_K2520036, BBa_K2520037) is a trans-membrane domain that anchors all the components located between the Igk leader and the PDGFR itself to the membrane. HA (BBa_K2520030), Myc (BBa_K2520031) and His (BBa_K2520032) are tags that bind to specific antibodies and provide an indirect method for verifying the display of the proteins on the membrane. The proteins that we chose to express on the membrane are three epitopes that are known targets of multiple sclerosis (BBa_K2520038, BBa_K2520039, BBa_K2520040) and are components related to our specific project. P2A (BBa_K2520033) and T2A (BBa_K1537017) are internal peptide cleavage sites that allow for the equimolar expression of multiple proteins under the same promoter.
The CMV promoter (BBa_K1119006) is a constitutive expression promoter in mammalian cells and hGH (BBa_K404108) serves as a terminator.
Structure
The structure of the Tri-Display components is shown below.
Characterization
We characterized this device by transfecting HEK-293 cells with this device, in comparison to non-transfected cells (NT). We incubate the transfected cells with specific antibodies that bind to three different tags (HA, Myc and His) conjugated to chromophores and measured the fluorescence intensity emitted (Figure 2).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 614
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 679
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2264
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