Part:BBa_K4829017
IVT of this sequence produces mRNA coding for a dAb blocking PD1
This composite biobrick is one of multiple combinations possible using our modular mRNA-based protein expression systems. We have not produced this using IVT. However, considering our success with BBa_K4829003, we can confidently say that this will produce the required dAb. We would like to emphasise further, that though this composite bio brick has used the albumin(HSA) signal peptide, we urge the users of the dAb for this application(mRNA protein production) to try out various different signal peptides
Usage and Biology
- It has a T7 promoter (BBa K4829000), a 5'UTR(BBa K4829004), a coding sequence (which has a signal peptide(BBa K4829001) along with the dAb(BBa_K4829011), a 3'UTR(BBa K4829005), and a polyA tail(BBa K4829006).
- On In Vitro Transcription, followed by purification, the mRNA produced can be used to transfect HeLa cells with Lipofectamine messengerMax. After 6 hours, the protein will be detected in the supernatant.
- We recommend that anyone using this construct use modified nucleotides: N1-Methylpseudouridine, and cap the mRNA with CleanCapAG, to improve translational efficiency and reduce immune response to the mRNA.
This specific dAb is expected to be a neutralising one. We have not used it for neutralising purposes ourselves yet. However, the potential use of this dAb would be to block PD1 either in disease processes or possibly even in cell biology assays/related studies. We have not included his tag/FLAG tag in this sequence. However, it would be prudent to do so, for purification purposes. This part has been introduced as a part of our mRNA-based therapeutic platform we are introducing on the biobricks registry. The sequence has been optimised for production by human cells. We therefore also encourage any users of this part to make a composite along the lines of BBa_K4829003 to use this and characterise it further.
Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of activated T cells, B cells, and other immune cells. It plays a critical role in regulating immune responses, particularly in the context of preventing autoimmunity and controlling immune reactions in peripheral tissues.
Role of PD-1:
- Immune Regulation: PD-1 is a key negative regulator of T cell activation and function. Its main role is to prevent excessive immune responses and autoimmunity.
- Interaction with Ligands: PD-1 has two main ligands, PD-L1 (programmed death-ligand 1) and PD-L2. These ligands are expressed on a variety of cells, including tumour cells, antigen-presenting cells, and some non-immune cells in various tissues.
- Tumor Immune Evasion: Many tumours upregulate the expression of PD-L1, which binds to PD-1 on T cells, leading to the inhibition of T cell function. This mechanism allows tumours to evade immune surveillance.
The specific features of this dAb have been modelled by our dry lab team, and the details may be found on our dry lab page. A brief summary of the details is mentioned below.
The image shown here is of the model of the dAb, predicted using Alphafold
Figure 1. PD1(red) binding to our dAb(green)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
The functional parameters we have been able to measure are the Gibbs free energy of binding and the Kd of the interaction with PD1. These details have been obtained using the GRAMM software. A full and detailed explanation of how this tool was used may be found on our dry lab page, and we encourage any users of this part to go through the extensive documentation there. We will post a few images of the binding parameters obtained below:
Figure 2. The binding parameters obtained from the GRAMM software
Figure 3. The modelling of the dAb using alphafold2
Figure 4: The structure of the RNA of the CDS produced on IVT of this sequence. 'Loss' function value (AUP) = 131.75200000000004
Scale of the AUP
For more information on the AUP, please check out out pages of BBa_K4829019 onwards
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