Materials and Reagents

1. Glass coverslips (VWR International, catalog number: 16004-094 )
2. 8-well chamber frame (Nunc Lab-Tek chamber, Thermo Fisher, catalog number: 155411PK )
3. Six-well plate (NEST, catalog number: 703001 )
4. Sfp phosphopantetheinyl transferase (NEB, catalog number: P9302S )
5. Coenzyme A (CoA)-488 (NEB, catalog number: S9348 )
6. 4,5-bis(1,3,2-dithiarsolan-2-yl)resorufin (ReAsH-EDT2, Santa Cruz, catalog number: sc-391916 )
7. TCEP (tris(carboxyethyl) phosphine) (Sigma, catalog number: C4706 )
8. X-tremeGENE HP DNA Transfection Reagent (Roche, catalog number: 0 6366236001 )
9. MES (2-mercaptoethanesulfonate) (Macklin, catalog number: S818461 )
10. BAL (2,3-dimercaptopropanol) (Macklin, catalog number: D807026 )
11. Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Human IgM, Fc5μ fragment specific (Jackson ImmunoResearch, catalog number: 109-607-043 )
12. HBSS (Hank's Balanced Salt Solution) (Gibco, catalog number: 14025092 )
13. Zeba Spin Desalting Columns (Thermo Fisher, catalog number: 89882 )
14. SYLGARD^TM 164 Silicone Elastomer Kit 210 ML KIT (Dow Corning, catalog number: 4028273 )
    

Equipment

1. Fluoview FV1000 Laser Scanning Confocal Microscope (Olympus)

Procedure

1. Site-specific labeling of VRC01-IgM-BCR expressed in 293T cell
   1. The constant region of human IgM heavy chain was fused with VRC01-specific variable region of heavy chain to construct mIg heavy chain of VRC01-mIgM-BCR (pHAGE-VRC01-H), while human Igκ constant region was fused with VRC01-specific variable region of light chain to construct mIg light chain of VRC01-mIgM-BCR (pHAGE-VRC01-L).
   2. A similar strategy was used for the construction of Ig heavy chain and Ig light chain of soluble VRC01-IgM. In addition, His₆ tag was fused at C terminus of Ig heavy chain of soluble VRC01-IgM for protein purification (pHAGE-VRC01-H-His6).
   3. ybbR tag and tetracysteine tag were inserted in plasmid carrying mIg heavy chain of VRC01-IgM-BCR to construct dually tagged VRC01-mIgM-BCR (pHAGE-VRC01-H-Tag), while ybbR tag and tetracysteine tag were inserted in plasmid carrying Ig heavy chain of soluble VRC01-IgM to construct dually tagged soluble VRC01-IgM (pHAGE-VRC01-H-His6-Tag). Insertion sites for the two tags are indicated in bold in the following table (Table 1).
      All the plasmids are not commercially available. Please find the construction strategy illustrated in Figure 1. The DNA sequence of VRC01 variable region, ybbR tag, and tetracysteine tag are listed in Table 2. All the plasmid constructions were performed following Gibson assembly protocol (Gibson et al., 2009). Protein sequences are listed in Table 3.
      
      
      Figure 1. Schematic figures of the construction strategies on mIgM-BCR structure and tagged VRC01-mIgM-BCR/soluble VRC01-IgM construction
      
      Table 1. Insertion sites for the ybbR tag or tetracysteine tag in IgM-BCR
      
      
      Table 2. DNA sequences for plasmids construction
      
      
      Table 3. Amino acid sequences of the constructions
      
      
      
      
2. An overall scheme of site-specific labeling procedures can be found in Figure 2.
   
   
   Figure 2. Overall scheme of site-specific labeling procedures for VRC01-IgM-BCR expressed in 293T cells
   
   
   1. Plate 293T cell in 6-well plate with DMEM containing 10% FBS and 1% penicillin-streptomycin reaching 50-60% confluency.
   2. For each 35-mm well in a six-well plate, prepare one vial of transfection medium containing 15 μl of X-tremGENE transfection reagent and 3.5 μg of the pHAGE-VRC01-H/pHAGE-VRC01-H-Tag, 2.5 μg pHAGE-VRC01-L, 2.5 μg pHAGE-CD79A and 2.5 μg pHAGE-CD79B plasmid diluted in 300 μl of opti-MEM (1×).
   3. Mix the transfection medium well and allow it to come to equilibrium for 30 min at room temperature.
   4. Incubate the cells with transfection medium for 5 h, and then incubate the cells in DMEM medium (1×) with penicillin and streptomycin for 24 h to allow time for protein expression.
   5. For ybbR tag labeling, detach the cells expressing dually tagged or untagged VRC01-IgM-BCR with trypsin and incubate them with 1 μM SFP Synthase, 2 μM CoA 488 and 10 mM MgCl₂ in 500 μl HBSS for 20 min at room temperature, then wash the cells with HBSS.
   6. Labeling of tetracysteine tag in cells is adopted from published protocols (Hoffmann et al., 2010). Pre-incubate the cells in HBSS containing 5 mM MES and 0.5 mM TCEP for 20 min at room temperature, then wash with HBSS and stain 1 mM ReAsH-EDT2 and 25 mM 2,3-dimercapto-1-propanol in 500 μl HBSS for 10 min at 4 °C. After staining, wash the cells with HBSS containing 100 mM BAL.
   7. Stain the cells with 100 nM Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Human IgM Fc5μ for 5 min at 4 °C in 500 μl HBSS, then wash twice with HBSS. Cells are ready for imaging.
   
   
3. Site-specific labeling of soluble VRC01-IgM
   1. 293F cells were co-transfected with plasmid carrying dually tagged (pHAGE-VRC01-H-His6-Tag) or untagged Ig heavy chain of VRC01-IgM (pHAGE-VRC01-H-His6) and mIg light chain of VRC01-IgM (pHAGE-VRC01-L).
      Note: Please refer to B1 to B4 for transfection protocols.
   2. Ectopically express and purify the IgM protein in HEK293F cells according to standard protocols (Portolano et al., 2014).
   3. For ybbR tag labeling in soluble VRC01-IgM, purified proteins were incubated in HBSS containing 1 mM SFP synthase, 10 mM MgCl₂, 5 mM CoA 488, 10 mM HEPES for 30 min at 37 °C. Then the labeled proteins were desalted using Zeba Spin Desalting Columns.
   4. For tetracysteine tag labeling, soluble VRC01-IgMs were treated with 1 mM ReAsH-EDT2 in HBSS at room temperature for 30 min and were desalted using Zeba Spin Desalting Columns according to standard protocol. In short, after removing the column’s bottom closure and loosen cap, centrifuge at 1,500 × g for 1 min to remove storage solution. Slowly apply 30-130 µl of the protein sample to the center of the compacted resin bed, followed with centrifuge at 1,500 × g for 2 min to collect the desalted sample.
      
      
      
4. Imaging of labeled VRC01-IgM-BCR expressing 293T cells or labeled soluble VRC01-IgM
   1. Chamber coverslip was fabricated according to our previous publication (Wang et al., 2018). In short, the coverslip was pretreated with piranha buffer (H₂SO₄:H₂O₂ = 7:3) for overnight, after extensively washing with ddH₂O, the coverslip was glued to the 8-well chamber frame with SYLGARD^TM 164. After curing, the chamber coverslip was ready to use.
   2. Labeled 293T cells expressing dually tagged or untagged VRC01-IgM-BCR were resuspended in HBSS and then loaded on chamber coverslip.
   3. Labeled dually tagged or untagged soluble VRC01-IgMs were loaded on coverslip for 30 min at 4 °C to allow the adherence on the surface. Then soluble VRC01-IgMs were stained with 100 nM Alexa Fluor 647 AffiniPure Fab Fragment Goat Anti-Human IgM Fc5μ in HBSS for 10 min at 4 °C, followed by washing with HBSS.
   4. Confocal images were acquired by Fluoview FV1000 Laser Scanning Confocal Microscope with 60× 1.42 NA oil objective lens. Typical images were shown in Figure 3.
   5. All the images were analyzed with ImageJ.
      
      
      Figure 3. Site-specific labeling in mIg heavy chain of IgM-BCR. Representative confocal images of tagged (A) and untagged VRC01-IgM-BCR (B) expressed in 293T cells. ybbR tag and tetracysteine tag were labeled by CoA 488 and ReAsH, respectively. Alexa Fluor 647 (AF647) Fab fragment of goat anti-human IgM Fc5μ was used for IgM-BCR staining. Scale bars = 10 μm.

Acknowledgments

This work has been supported by funds from National Natural Science Foundation of China (81825010, 81730043, 81621002, 81961130394 and 31811540397). It is also supported by Beijing Advanced Innovation Center for Structural Biology, Center for Life Sciences, and Institute for Immunology at Tsinghua University. This protocol was derived from previous paper published in eLife (Shen et al., 2019).

Competing interests

The authors declare no financial or commercial conflict of interest.

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