Introduction
We are going to work with 2 different proteins, each having the normal form and the extended form (which contains an additional sequence in the middle). The first protein is going to be named "Protein A" and the second one "Protein B". At the end we will have:
- Prot A normal → referred as 1
- Prot A extended → referred as 2
- Prot B normal → referred as 3
- Prot B extended → referred as 4
Protein names are censored due to pending patents, and therefore cannot be disclosed at this stage. Once the intellectual property protection process is completed, the specific protein identities will be available.
Following we explain the additional parts that were added in the plasmid TrcHisB. At the end we obtained 4 gblock sequences due to the fact that we have four protein constructs, two for protein A and two for protein B. The other parts were the same in all cases.
The part we created contains: 6HIS - GST - TEV site - Protein - T7 tag
The inclusion of both His and GST tags allows purification using either tag individually or in combination, which increases the purity of the final protein preparation.
Plasmids
| ID Code | Name | Type | Description | Notes / Usage | Part |
|---|---|---|---|---|---|
| BBa_J96037 | His | Protein domain | Is a 6-histidine tag, used for rapid purification using nickel columns and can also aid in detection. | Present in all 4 constructs | Registered |
| BBa_25XVGUCE | GST tag | Protein domain | Allows for affinity purification using glutathione beads, it can also be used in the aptamer SELEX for the selection of the sequences of interest. | Present in all 4 constructs | New part |
| — | Protein A construct | Composite part | Construct encoding Protein A fused to: 6xHis – GST – TEV site – Protein A – T7 tag | Used as protein of interest | — |
| — | Protein A Extended construct | Composite part | Construct encoding Protein A (extended version) fused to: 6xHis – GST – TEV site – Protein A extended – T7 tag | Designed to compare structural and functional differences with Protein A | — |
| — | Protein B construct | Composite part | Construct encoding Protein B fused to: 6xHis – GST – TEV site – Protein B – T7 tag | Used as protein of interest | — |
| — | Protein B Extended construct | Composite part | Construct encoding Protein B (extended version) fused to: 6xHis – GST – TEV site – Protein B extended – T7 tag | Designed to compare structural or functional differences with Protein B | — |
| BBa_25TB01I3 | TEV site | Scar | Is a sequence recognized by the TEV protease. It is used to cut and remove purification tags from the protein once it has been isolated. | Present in all 4 constructs | New part |
| BBa_K5261011 | T7 tag | T7 | Is a small peptide derived from bacteriophage T7. It is used as an epitope tag to detect the protein using anti-T7 antibodies. | Present in all 4 constructs | Registered |
Protein
Protein A
Protein B
