We intend to create a cellular hemoglobin (Hb) based oxygen carriers for future artificial blood design. To reach this goal, we firstly need to find Hb and then found proper carrier. We researched and found hemoglobin isolated from Vitreoscilla (VHb) is an ideal hp to start our project, VHb is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions. Fortunately the part with VHb is available in distribution kit with the name of BBa_K1321200.
We improved this part by creating a composite part CD63-Vhb Fusion Protein (BBa_K2794005), when this fuse genes been transfected into mammalian cells, the fusion protein will be transported to the endosome and then through inward budding and exocytosis be exported on the membrane of exosomes. In this way, Vhb located inside the exosome and it automatically transported into the exosomes.
We constructed plasmids pRK7-N-Flag-Vhb (abbreviated as PNV) and pRK7-N-Flag-CD63-Vhb plasmid (abbreviated as PNCV) to express the target protein in HEK 293T cells. We have sequenced the plasmid and confirmed the sequence. Then we did a series of experiments to study the function of the fusion protein
Whole cell lysate western blotting result showed successful transfection of PNV and PNCV plasmids in HEK 293T cells. Exosome content showed successful result of fusion protein band around 40 kDa. TEM result of sample shown that the protein doesn’t effect the shape or the concentration of exosomes. COD result compared to normal exosomes shown that the fusion protein contains heme group that increases the chemical demand of exosomes and thus proving the function of Vhb is not disturbed. Also, color difference can be seen between normal and fusion-protein containing exosomes.