Nanobody screening by phage display

Protocol for Amplification of Phage-Displayed Nanobody Library

The stock vial we distributed contains 1.6 mL of phage glycerol stock of the nanobody library, with a titer of 2.2 × 10¹¹ cfu/mL. The stock should be stored at -80°C. The diversity of the nanobody library is 2.4 × 10¹⁰. To maintain the integrity of the library during amplification, and considering that E. coli reaches a density of approximately 5 × 10⁸ cells/mL at OD600 ~0.6, we recommend using at least 200 mL of bacterial culture for phage infection during amplification.

Material

• 2×YT Medium (Tryptone 16 g/L, Yeast extract 10 g/L, NaCl 5 g/L. Autoclave at 121 °C for 30 min.)
• PEG/NaCl solution (20% PEG-8000 (w/v), 2.5 M NaCl. Autoclave at 121 °C for 30 min.)
• Tetracycline (prepared as 1000× stock, final concentration in 2×YT medium is 10 μg/mL)
• Ampicillin (prepared as 1000× stock, final concentration in 2×YT medium is 100 μg/mL)
• Kanamycin (prepared as 1000× stock, final concentration in 2×YT medium is 50 μg/mL)
• OmniMAX E. coli cells (Invitrogen, C854003)
• M13KO7 helper phage (Invitrogen, 18311019. You can also use your homemade M13KO7 helper phage, but make sure the titer of M13KO7 is >1×10¹¹)

Protocol

1. Thaw a tube of OmniMax bacteria on ice and inoculate it into 200 mL of 2×YT medium without antibiotics. Incubate at 37°C with shaking at 220 rpm for 1 hour. Then add tetracycline to the medium and continue culturing until the OD600 reaches approximately 0.6.

   Note: A 1-hour culture in 2×YT medium without antibiotics allows for the expression of the antibiotic resistance gene.

2. Let the bacteria stand for 5 minutes to allow regeneration of the sheared F pili.

3. Add 200 µL of phage glycerol stock to the 200 mL bacterial culture and incubate at 37°C with shaking at 220 rpm for 20 minutes to allow phage infection.

   Note: Based on the titration, the 200 µL phage glycerol stock contains approximately 4.4 × 10¹⁰ transducing units. The 200 mL bacterial culture at OD600 ~0.6 contains roughly 1 × 10¹¹ cells, resulting in a multiplicity of infection (MOI) of less than 0.5.

4. Take 10 µL of the infected bacterial culture and perform serial dilutions to estimate the number of infected bacteria.

   Note: The number of infected bacteria should exceed the diversity of the library to ensure the integrity of the library during amplification.

5. Add 20 mL of M13KO7 helper phage (titer > 1 × 10¹¹) to the culture and incubate at 37°C with shaking at 220 rpm for 40 minutes.

   Note: The 20 mL of M13KO7 helper phage contains approximately 2 × 10¹² infectious particles. The 200 mL bacterial culture at OD600 ~0.6 contains roughly 1 × 10¹¹ cells, resulting in a multiplicity of infection (MOI) of approximately 20.

6. Transfer the culture into 2000 mL of 2×YT medium supplemented with ampicillin and kanamycin, and incubate overnight at 37°C with shaking at 220 rpm.
7. Centrifuge to collect the supernatant.
8. Precipitate phage from the supernatant using PEG/NaCl and resuspend in PBS. (Phage precipitation was performed in 50 mL tubes, each containing 36 mL of bacterial supernatant. Add 9 mL of PEG/NaCl solution to each 36 mL supernatant, then incubate at 4°C for 2 hours. Centrifuge at 12,000 × g for 30 minutes. Resuspend the resulting pellet in 2 mL of PBS per tube.)
9. Centrifuge at 12,000 × g for 3 minutes to remove undissolved material.

10. Titrate the phage solution by performing 10-fold serial dilutions. Mix 10 µL of each diluted phage solution with 100 µL of OmniMax cells at OD600 ~0.6, incubate at room temperature for 20 minutes, then spread on ampicillin LB plates. Count colony number after overnight culture.

    Note: Normally the titer is over 1×10¹¹.

11. Mix the phage solution with an equal volume of 80% glycerol (1:1) to prepare the glycerol stock of the phage library. Aliquot 1.6 mL of the glycerol stock into each 2 mL tube. Each tube is sufficient for one screening campaign.

Protocol for Nanobody Screening with Phage-Displayed Nanobody Library

Materials

• Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific, 442404)
• Target proteins
• Control proteins (bearing the same tags as the target proteins, used to deplete nanobodies that bind the tags)
• PBST (PBS + 0.05% (v/v) Tween 20)
• MPBST (5% skim milk in PBST, freshly prepared)

Protocol

Coat Nunc MaxiSorp 96-well plates with antigen:

Negative selection: Coat one Nunc MaxiSorp 96-well plate with control proteins at 1 µg/well for each round. Incubate overnight at 4 °C.

Positive selection: Coat one Nunc MaxiSorp 96-well plate with target proteins at 1 µg/well for the first round, 0.5 µg/well for the second round, and 0.25 µg/well for the third round. Incubate overnight at 4 °C.

Nanobody screening:

1. Inoculate OmniMAX bacteria into 6 mL 2×YT medium containing tetracycline.

   Note: The selection procedure takes 4-5 hours. Adjust the bacteria amount so that the bacteria reach OD600 ~0.6 in 4-5 hours.

2. Block both the negative and positive selection plates with 5% MPBST at room temperature for 1-2 hour with shaking at 600 rpm. 

   Note: Completely fill each well (~200 µL per well).

3. Thaw one tube of phage library (1.6 mL) and mix it with 4.5 mL of 5% MPBST.
4. Remove the blocking buffer from the negative selection plate. Add 60 µL/well of the phage solution and incubate at room temperature for 1 hour with shaking at 600 rpm.

5. Remove the blocking buffer from the positive selection plate. Transfer the phage solution from the negative selection plate to the positive selection plates. Rinse the negative selection plate with 90 µL 5% MPBST per well and transfer the rinse to the positive selection plates, bringing the final volume in each antigen-coated well to 150 µL. Incubate at room temperature for 1 hour with shaking at 600 rpm.

   Note: This step minimizes phage library loss during transfer.

6. Wash the positive selection plate 10 times with TBST.

   Note: Wash the positive selection plate 20 times with TBST for the second round and 30 times for the third round.

7. Add 50 µL OminiMAX bacteria at OD600 ~0.6 to each well and incubate at room temperature for 20 min with shaking at 600 rpm to allow phage infection. 
8. Collect all bacteria and add helper phages at 1/10 of culture volume (MOI ~20). Incubate at 37°C with shaking at 220 rpm for 40 minutes. 
9. Transfer the culture into 36 mL of 2×YT medium containing ampicillin and kanamycin, and incubate overnight at 37°C with shaking at 220 rpm.
10. Precipitate phages from the supernatant using PEG/NaCl method. Dissolve the phage pellet using 2 mL PBS. Use 1 mL phage solution for the next round of selection.