Method of purifying authentic trimeric HIV-1 GP140 envelope glycoproteins comprising a long linker and tag
An approach of producing recombinant trimers that mimic native HIV-1 envelope trimers is developed. A recombinant protein forming the recombinant trimers encompasses a recombinant HIV-1 gp140 fused to a tag through a linker at C-terminus of the recombinant HIV-1 gp140. The linker is sufficiently long so that the tag is accessible for binding by a binding molecule bound on a solid matrix. After expressed in a cell, the recombinant protein is secreted into the culture medium and assembles into recombinant trimers therein. The recombinant trimers may be directly purified from the culture medium. Cleaved and uncleaved trimers from different clade viruses are produced.
1. A method comprising:
binding a recombinant HIV-1 envelope trimer contained in a culture medium to a binding molecule immobilized on a solid matrix; and
eluting the recombinant HIV-1 envelope trimer that binds to the binding molecule immobilized on the solid matrix with an elution buffer, thereby producing a purified recombinant HIV-1 envelope trimer,
wherein the recombinant HIV-1 envelope trimer is formed by a recombinant protein comprising a recombinant HIV-1 gp140 attached to a tag through a linker at C-terminus of the recombinant HIV-1 gp140,
wherein a base of the recombinant HIV-1 envelope trimer mimics a native HIV-1 envelope trimer,
wherein the linker allows a formation of the base of the recombinant HIV-1 envelope trimer,
wherein the linker is not required for the formation of the base of the recombinant HIV-1 envelope trimer,
wherein the linker has a sufficient length to separate the tag from the base of the recombinant HIV-1 envelope trimer so that the binding molecule immobilized on a solid matrix is accessible to the tag without steric hindrance, and
wherein the linker comprises at least 20 amino acids.
2. The method of claim 1 , wherein the recombinant protein comprises a gp120 and a gp41 ectodomain connected by junction sequence comprising a furin cleavage resistant sequence, and wherein the recombinant HIV-1 gp140 is uncleaved.
3. The method of claim 2 , wherein the linker comprises at least 23 amino acids.
4. The method of claim 2 , wherein the linker comprises SEQ ID NO: 1.
5. The method of claim 2 , wherein the linker comprises SEQ ID NO: 5.
6. The method of claim 2 , wherein the linker comprises SEQ ID NO: 6.
7. The method of claim 2 , wherein the linker comprises SEQ ID NO: 7.
8. The method of claim 2 , wherein the linker comprises a flexible linker.
9. The method of claim 8 , wherein the flexible linker comprises SEQ ID NO: 8.
10. The method of claim 2 , wherein the linker comprises a rigid linker.
11. The method of claim 10 , wherein the rigid linker comprises SEQ ID NO: 9.
12. The method of claim 2 , wherein the recombinant HIV-1 gp140 comprising a truncation at aa664 based on HXB2 numbering.
13. The method of claim 2 , wherein the recombinant HIV-1 gp140 comprises “SOSIP” mutations that comprise A501C, T605C, and I559P, and wherein the recombinant HIV-1 gp140 comprises five “stabilizing” mutations comprising I535M, Q543L, S553N, K567Q, and R588G.
14. The method of claim 2 , wherein the recombinant HIV-1 envelope trimer that binds to the binding molecule immobilized on the solid matrix is separated from the culture medium by centrifugation.
15. The method of claim 2 , wherein the binding step is carried out by affinity chromatography, and size exclusion chromatography is conducted after eluting the recombinant HIV-1 envelope trimer that binds to the binding molecule immobilized on the solid matrix with an elution buffer.
16. The method of claim 2 , wherein the tag comprises SEQ ID NO: 2, and wherein the binding molecule immobilized on a solid matrix comprising a Strep-Tactin bead.
17. The method of claim 2 , wherein the tag comprises SEQ ID NO: 3, wherein the binding step is carried out by HisTrap affinity chromatography and by size exclusion chromatography, and wherein the size exclusion chromatography is conducted after the HisTrap affinity chromatography is conducted.
18. The method of claim 2 , wherein the recombinant protein is expressed by a cell growing in the culture medium, wherein the cell growing in the culture medium is transfected with a recombinant DNA encoding the recombinant protein, wherein a secretion signal peptide is fused at N-terminus of the recombinant protein, and wherein the recombinant protein is secreted into the culture medium when the recombinant protein is expressed by the cell.
19. The method of claim 18 , wherein the cell comprises a 293F cell.
20. The method of claim 18 , wherein the cell comprises a GnTi − cell.