Recombinant Filovirus Glycoprotein (GP) Trimer

Background

The filovirus family includes Marburg virus and five ebolaviruses (Ebola-, Sudan-, Reston-, Bundibugyo- and Taï Forest viruses). Filoviruses express a single protein on their envelope surface, a glycoprotein termed GP, which is responsible for attachment to, and entry of, host cells. GP forms a trimer on the viral surface. In the trimer, each monomer comprises GP1 and GP2 subunits that are anchored together by a GP1-GP2 disulfide bond. GP1 contains a receptor-binding core topped by a glycan cap and a heavily glycosylated mucin-like domain, mediating receptor binding, while GP2 contains two heptad repeats and a transmembrane domain, facilitating viral membrane fusion to initiate infection. The trimeric fusion GP is the sole target for neutralizing antibodies and is the major focus of vaccine development.

Fig. 1 Sequence and structure analysis of Filovirus GP glycosylation

Challenges in Producing Recombinant Filovirus GP Trimers

Expression of filovirus GP trimers presents specific obstacles. The mucin-like domain and extensive glycosylation dramatically reduce expression efficiency and yield of the full-length GP, often leading to multiple heterogeneous species during purification. Deletion or truncation of the mucin-like domain is frequently used to increase expression without severely compromising key neutralizing epitopes.

In addition, the prefusion trimer is intrinsically unstable; without rationally introduced stabilizing mutations in the hinge or interface regions, GP tends to transition toward postfusion or partially unfolded states, diminishing its antigenicity.

Finally, host cell selection and downstream purification require careful optimization to support complex glycan processing, furin cleavage, and removal of misprocessed or co-purified contaminants that otherwise impair purity and batch-to-batch consistency.

Recombinant Filovirus GP Trimer Proteins at Creative Diagnostics

Creative Diagnostics has successfully developed several trimeric Filovirus GP proteins for better support of Filovirus vaccine and drug development. These HA trimer proteins are produced in HEK293 mammalian cells, ensuring proper folding, trimerization, and mammalian-like glycosylation. High-purity preparations are confirmed by SDS-PAGE, demonstrating the correct size and integrity of the expressed trimer.

SDS-PAGE

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