Cell surface IgM expression of PGT128/130 gl precursor

To enable cell surface expression of the predicted PGT128/130 gl antibody precursor²⁰ in its IgM form, the 3′ end of the gene encoding soluble IgM in vector pFUSEss-CHIg-hM*03 (Invivogen) was replaced with that of membrane-bound IgM (mI gM)⁴⁷ to yield vector pFUSEss-CHIg-mhM*03v2. The V_H segment of the gl precursor was then subcloned into the resulting vector using as PCR template a plasmid encoding the heavy chain of the antibody (kindly provided by Dennis Burton). The resulting heavy-chain-expressing plasmid was then mixed with the transfection reagent Fugene HD (ThermoFisher) at a 1:1 ratio with a light-chain expression plasmid (also kindly provided by Dennis Burton) and transfected into mycoplasma-free 293T cells (ATCC; cat. no. CRL-11268). Cells were trypsinized (TrypLE Express; ThermoFisher) 3 days post-transfection, and re-suspended in FACS staining buffer (Hank’s Buffer Staining Solution (HBSS; Lonza) supplemented with 10% (v/v) FBS (Thermo Fisher)). The cells were then incubated with NIT150b, a biotinylated version of the NIT82B glycoconjugate (see above), and then with phycoerythrin-conjugated streptavidin and allophycocyanin-conjugated anti-human F(ab’)₂ (both from Jackson ImmunoResearch). All incubation steps were performed for 30 min on ice and samples washed and re-suspended in staining buffer between steps. The data were acquired on a BD FACSJazz and analyzed with FlowJo software (v10; Treestar).