Project Overview

Blood donations are needed all around the world. Due to the fact that humans have at least four different blood groups and two different Rhesus factors - all antigens on erythrocytes - blood can not simply be transfused to any random patient. Only blood type O RhD(-) is the universal transfusion blood type, which can be received by every person. One exception is the rare Bombay type. Because of its universal applicability, blood group O is generally used as emergency donor blood, in cases where a patient urgently needs transfusion.

This is where we came up with and iea for a practical system to convert blood groups of type A, B and AB of the ABO blood group system into blood group O. The antigens of the ABO blood group system consist of glycosylated proteins on the surface of erythrocytes. Antigens of blood group A and B both have a distinct glycosylation. Blood group AB has both antigens present on the erythrocyte surface. The antigen of blood group O only consists of the primitive glycosylation of five sugars, this is the reason why blood type A and B cause no immunoreaction. Bombay type blood (Oh) has even less sugar residues. Therefore, persons with the Bombay type blood can only accept Bombay type blood.

The main goal of our project is the covalent immobilization of three conversion enzymes onto a matrix. These enzymes are N-Acetyl-Galactosaminidase (NAGA) from Elisabethkingia meningosepticum , α-Galactosidase (aGAL) from Bacteriodes fragilis and Endo - β - galactosidase (EABase) from Clostridium perfringens. NAGA removes N-Acetylgalactosamin (GalNAc) from erythrocytes and therefore converts blood group A to O and blood group AB to B. aGAL removes Galactose and thus converts blood group B to O and blood group AB to A. EABase converts blood group A, B and AB to Bombay type. For cloning, we used different E. coli strains: NEB5α, BL21(DE3) and XL10 Gold. For the immobilization on agarose beads three different tags are used.

Figure: Schematic illustration of the enzymatic action of NAGA, aGAL, and EABase. While NAGA cleaves blood type A antigens, aGAL cleaves blood type B antigens. Both create type O antigens. EABase, however, cleaves both A and B antigens in a way that creates the so called Oh antigens - which corresponds to a blood type known as "Bombay".

The first tag is called SNAP-tag and is coupled with the enzyme. They are expressed together as one protein chain and then the SNAP-substrate is fused to agarose beads. The second tag, named SpyTag, consists of two parts: the SpyCatcher, which is coupled with the enzyme, and the SpyTag, which is a synthetic peptide attached to a membrane. Both parts are fused together, forming an immobilized enzyme. The third tag is called Ssp GyrB split intein (intein-tag) and is derived from a self-splicing protein. When both parts approach each other, the intein parts fuse and splice themselves, leaving behind an immobilized enzyme on the membrane.

For the analysis, a simple clotting test with blood antibodies (commonly used to identify a patient's blood group) will be used for the determination of the activity of the soluble enzymes and FACS for the immobilized enzymes' activity, using special fluorescence coupled antibodies.

Methods used during the practical part varied from restriction, ligation and transformation over agarose gelelectrophoresis, SDS page and PCR to gelextraction, plasmid preparation and peptide synthesis.