Method for producing erythrocyte proteins
The invention relates to a novel method for the synthesis of an erythrocyte protein, in which said protein is synthesized in an acellular system for the production of proteins, in the presence of at least one detergent which is non-ionic, of liposomes or of nanodiscs. The invention also relates to compositions comprising the proteins produced in this way.
1. A method of synthesizing an erythrocyte protein in its native form selected from RhD of SEQ ID NO: 5, RhCE of SEQ ID NO: 6, RhAG of SEQ ID NO: 7 and UTB of SEQ ID NO: 8, or a variant thereof, said method comprising the steps of:
a) contacting a nucleic acid encoding said protein or a variant having at least 95% identity therewith with an acellular protein production system, in the presence of at least one non-ionic detergent selected from Brij-35 and Brij-58, liposomes or nanodiscs; and
b) synthesizing said protein,
wherein step a) occurs either during or before step b).
2. The method according to claim 1 , wherein the contacting according to step a) occurs in the presence of nanodiscs comprising a MSP protein selected from proteins of SEQ ID NO: 9, 10 or 11.
3. The method of claim 2 , wherein said MSP protein is fused to a tag sequence.
4. The method according to claim 1 , wherein the acellular system for protein production is a batch system or an acellular system with continuous exchange.
5. The method according to claim 1 , wherein the concentration in nanodiscs is less than 80 μM.
6. The method according claim 1 , wherein said erythrocyte protein, or variant thereof is fused to a tag sequence.
7. The method of claim 1 further comprising a step of recovering the erythrocyte protein or variant thereof.
8. The method of claim 1 further comprising a step of fixing the erythrocyte protein or variant thereof to a solid support.
9. The method of claim 1 , wherein said nanodiscs comprise of one or more phospholipids selected from among dioleoylphosphocholine, dimyristoylphosphocholine, and palmitoyl oleoyl-glycero-phosphocholine.
10. The method of claim 1 , wherein said non-ionic detergent is used at a concentration comprised between 0.1% and 5%.
11. The method of claim 1 , wherein said non-ionic detergent is used at a concentration of 0.5%.