Method of purifying authentic trimeric HIV-1 envelope glycoproteins comprising a long linker and tag
AssigneeThe Catholic University of America
InventorsVenigalla B. Rao, Wadad Alsalmi
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Abstract
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.
Claims24
A method comprising:
- purifying a recombinant HIV-1 envelope trimer of a heterodimer from a culture medium containing the recombinant HIV-1 envelope trimer via a binding molecule bound to a solid matrix to produce the recombinant HIV-1 envelope trimer,
- wherein the heterodimer comprises a cleaved gp120 and a cleaved gp41 ectodomain attached to a tag through a linker at C-terminus of the cleaved gp41 ectodomain,
- wherein the recombinant HIV-1 envelope trimer comprises a recombinant trimer base comprising the cleaved gp120 and the cleaved gp41 ectodomain not including the linker and the tag,
- wherein the linker is not required for the formation of the recombinant trimer base,
- wherein the linker has a sufficient length to separate the tag from the recombinant trimer base so that the binding molecule bound on a solid matrix is accessible to the tag without steric hindrance,
- wherein the recombinant trimer base mimics a native HIV-1 envelope trimer, and
- wherein the linker comprises at least 20 amino acids.
Dependencies are inferred from the claim text.