HIV gp120 Nanofiber Conjugation

Subtype C, CH505 strain consensus HIV Env gp120 antigen (CH505 Con gp120) was expressed with sortase enzyme recognition sequence (LPETG) on the C terminus (43 (link)). To conjugate CH505 gp120 to G₁₅–β tail peptide (G₁₅-MALKVELEKLKSELVVLHSELHKLKSEL), 10.9 μM CH505 gp120 was coincubated with 25 μM sortase A and 60 μM G₁₅–β tail overnight at 4°C (36 (link)). As sortase A was expressed with poly-histidine tag, we removed it from the mixture after conjugation reaction by using a His SpinTrap column (Cytiva Life Sciences, #28401353). The resultant CH505 gp120–β tail conjugate was mixed with 10 mM Q11 peptide (QQKFQFQFEQQ) aqueous solution in 4:1 ratio (v/v), yielding a final concentration of 2 mM Q11. Overnight incubation allowed coassembly of gp120–β tail and Q11 peptides into nanofibers (28 (link)). Nanofibers bearing conjugated gp120 were pelleted at 9000g to separate assemblies from unassembled proteins in solution, followed by gentle washing with phosphate-buffered saline (PBS) to remove nonspecifically bound protein.