His spintrap column

Cloning constructs were used to transfect FreeStyle 293-F cells that were grown in suspension using FreeStyle 293 expression medium (Life Technologies) at 37°C in a humidified 8% CO₂ incubator rotating at 125 rpm. Cells were grown to a density of 2.5 million cells per ml, transfected using PEI (4 μg/ml in cell suspension) and DNA (1200 ng/ml in cell suspension), and cultivated for 3 days. The supernatants were harvested and proteins purified by His SpinTrap columns according to manufacturer’s instructions (Cytiva, 95056-290). The eluted protein was transferred to phosphate-buffered saline (PBS) via buffer exchange using Amicon Ultra-4 ultrafiltration column with 50 kDa cut-off (Millipore, UFC805008). Protein concentration was determined by His-tag specific ELISA using a mouse anti-His-tag antibody (Abcam, #ab18184) and a goat anti-mouse IgG Fc antibody conjugated to alkaline phosphatase (Southern Biotech, #SBA-1033-04) as detection reagent. Protein production was confirmed by SDS-PAGE and western blot using a mouse anti-His antibody (Abcam, #ab18184) and an IRDye 800CW donkey anti-mouse antibody (Li-Cor Biosciences, #925-32212).

Purification was performed according to Cranfill et al. with minor modifications.^{36 (link)} Briefly, One Shot TOP10 Chemically Competent E. coli (Thermo Fisher, Cat# C404003) were transformed with pBAD/HisB-PAmKate (Plasmid #32691, Addgene)^{37 (link)} and grown to OD600 0.6 in LB medium containing 0.1 mg ml−1 ampicillin at 37 °C. Heterologous protein expression was then initiated with 0.02% w/v L-Arabinose (Sigma A3256) overnight at 30 °C. Cells were collected by centrifugation (4000x g, 10min) and frozen. For purification, cells were resuspended and lysed in BugBuster^® Master Mix Protein Extraction Reagent (Millipore Sigma, Cat# 71456) at room temperature following the manufacturers recommendation. Lysate was cleared by centrifugation (15,000x g, 10min) and the soluble fraction of His-tagged proteins was purified using His SpinTrap columns (Cytiva Cat# 28401353) following the manufacturer’s protocol. The eluate protein from NiNTA columns was buffer exchanged into PBS pH 7.4 and concentrated with a 3 kDa Amicon^® Ultra-4 Centrifugal Filter Units (Millipore Sigma Cat# UFC800308). Proteins were frozen and stored at −80 °C.

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.