Most artificial blood only focus on its ability to carry oxygen and its immune-compatibility. More specifically, for cellular hemoglobin based oxygen carriers, production of hemoglobin (Hb) and loading of Hb into its vascular structure.
The most commonly used carrier is the liposome, which is artificially made and can be loaded with protein using electroporation. In liposome based artificial blood, like LEAcHb, the surface of liposome need to be greatly modified using PEGylation or use monomeric actin to prolong its half life and immune-compatibility.
In our project, we first need to choose our Hb carrier. We first looked at previous attempts made by iGEM teams. In 2007, Berkley iGEM team created the BactoBlood, an artificial blood based on Hb, Heme and molecular chaperone containing E.coli. Although they used kill-switch and edited strain to prevent E.coli from causing immune problems, the bacteria still is far from use-ready for transfusion. We also thought about using bacterial outer membrane vesicle as our carrier, but it still can not solve the problem of bacterial antigen.
After some research, we’ve chosen mammalian exosomes as our Hb carrier. Exosomes are membrane vesicles 30nm-100nm in diameter and has specialized functions and play a key role in processes such as coagulation, intercellular signaling, and waste management, and has been already used by iGEM teams in RNAi targeted therapy. We’ve chosen exosomes because its immune-compatibility and it’s easiness to edit to produce.
Figure 1: Two methords of exosome cargo loading and formation of exosomes.
The major problem after that is how can we load protein cargo into exosomes. The commonly used method is to use electroporation to load proteins and RNA into it. But electroporation can greatly modify the morphology of exosomes and can sometimes be inefficient. Here, we will present the two new methods we’ve used to load the protein cargo: membrane protein anchoring and WW Tag induced active transportation of protein. And we will discuss how we constructed the plasmids for translation in mammalian HEK293T cells.
Our Hemoglobin: Vitreoscilla haemoglobin (BBa_K1321200)
We first tried to use human hemoglobin in ExoBlood, But we failed to get the required parts due to complexity, time and shipment difficulty. So alternatively we used Vitreoscilla haemoglobin (Vhb), which is a kind of hemoglobin used by multiple iGEM teams to increase bacterial survival in low oxygen environments, and is proven to bind heme and carry oxygen. So we subcloned Vhb from BBa_K1321200 in this year's distribution kit and used it for further construction of plasmids.
Figure 2: Structure of Vhb dimer and heme contained in it.
Methord to Load Cargo into ExosomesMembrane Protein Anchoring-CD63
CD63 is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. The proteins mediate signal transduction events that play a role in the regulation of cell development, activation, growth, and motility. This encoded protein is a cell surface glycoprotein that is known to complex with integrins. It may function as a blood platelet activation marker.
CD63 (varient 1) passes through the membrane 4 times. The N- and C- terminus is both located inside the membrane. The protein has two extracellular cringle (EC1, EC2) and one intracellular loop (IL), with EC2 significantly longer than EC1 and carries main cellular functions.
Figure 3: Structure of human tetraspanin CD83, similar to CD63
CD63 is recently researched and used in the field of extracellular vesicle (EVs) research. Due to its strong bond on EVs, it is used in exosomes identification, extraction and targeted therapy. A protein sequence can be attached to either the N- or C- terminus so that a protein cargo can be loaded into exosomes. This method is refereed as membrane protein anchoring.
Figure 4: Membrane Protein Anchoring Ndfip1-WW Tag Induced Active Cargo Loading Based on Ubiquitination and ESCRT Pathway
Nedd4 Family Interacting Protein 1 (Ndfip1, synonym N4WBP5) is a membrane protein located on the Golgi apparatus and endosome. It's a evolutionarily conserved protein with three transmembrane domains. This protein is thought to be part of a family of integral Golgi membrane proteins.
As said in the name of the protein, ndfip1 interacts with protein Nedd4, a ubiquitin-protein ligase. Nedd4 has 3-4 WW domain, a carboxyl-terminal Hect (homologous to E6-APCarboxyl terminus) domain and in most cases an amino-terminal C2 domain. Ndfip1 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein.
Figure 5: Structure of Nedd5
E3 ubiquitin ligase Nedd4 binds its WW domain to the PPXY motif on Ndfip1, and the cargo that also has ww domain binds to another PPXY motif on Ndfip1.
1: E3 ubiquitin ligase Nedd4 binds its WW domain to the PPXY motif on Ndfip1, and the cargo that also has ww domain binds to another PPXY motif on Ndfip1.
2: Ndfip1, as E3 ubiquitin ligase, transfer the ubiquitin to the tagged protein.
3: The ubiquitinated cargo is loaded into the endosome by the ubiquitination related endosomal sorting pathway ESCRT.
Figure 6: Active loading induced by WW tag and Ndfip1
Hence, co-expression of Ndfip1 and WW tag labeled protein can actively ubiquitinate target protein and made it loaded into exosomes and create exosomes for medical use. We’ve chosen the sequence of WW domain 3 and WW domain 4 on Nedd4 as our WW Tag.
Cells: HEK 293T
We used Human embryonic kidney 293T cells as our mammalian cell used in research. We’ve chosen HEK 293T because it grows very fast, and its a very regular cell used in exosome research. There are many papers related to the morphology of 293T exosomes and RNAi therapy research.
Figure 7: HEK 293T cells under microscope
We’ve used vector pRK7-N-Flag because it’s high expression, stability and easiness to experiment. The Flag Tag attached to the N terminus of the protein can be used in western blotting to prove the successful expression of proteins in cell and exosomes. HindIII, XbaI, EcoRI and BamHI sites in MCS is used.
Figure 8: Our designed plasmids.