Phage display

The term "phage display" describes an iterative process to select and enrich single antibodies out of trillions (10¹³) of clones with help of bacteriophages. Phages can infect bacteria, replicate and express the protein coded on dedicated vectors of the host. The advantage of phages is that they express the intact protein on their surface and enable binding to protein of interest. One round of phage display consisted of the production of phages, selection on the target protein, infection of bacteria with the selected phages and production of the selected nanobodies with subsequent screening or another round of phage display starting with infection of the bacteria with phages.

Phages are extremely volatile, thus the work with phages required special safety precautions such as special laboratories with dedicated fume hoods for phage work, use of filtered tips and decontamination of all contaminated material in bleach for at least 24 h.

Preparation of phages

50 ml 2YT medium supplemented with ampicillin (100 µg/ml) were inoculated with 25 µl of the antibody library or the output culture after each round of selection. The optical density at 600 nm (OD600) of the culture was 0.1 at the time of inoculation. The culture was incubated (37°C, 250 rpm) until OD600 > 0.4 and < 0.6. The OD600 must not exceed 0.6 because the culture had to remain in the exponential growth phase. The bacteria form pili during the exponential growth phase, which were needed by the phages for infection the bacteria. An OD600 of 1 represents 5 x 10⁸ bacteria per ml.

The culture was infected with an 20x excess of KM13 helper phage and incubated without shaking (30 min, 37°C). The culture was spun down (15 min, 4000 rpm, RT) and the pellet was resuspended in 250 ml 2YT medium supplemented with ampicillin (100 µg/ml) and kanamycin (50 µg/ml) and incubated overnight (30°C, 250 rpm). The following day, the culture was split and transferred in five 50 ml centrifugation tubes and spun down (20 min, 4000 rpm, 4°C). The supernatants were split in 25 ml fractions and transferred in clean 50 ml centrifugation tubes. Phages were precipitated by

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addition of 5 ml (1/5 of the volume) 20% PEG8000, NaCl 2.5 mM to each fraction and subsequent incubation on ice (1 h). The precipitated phages were spun down (15 min, 4000 rpm, 4°C), the supernatant was discarded and two pellets were pooled in 1 ml PBS, transferred in 1.5 ml Eppendorf tubes and spun (2 min, 14000 rpm, 4°C) to remove remaining bacteria. The supernatant was transferred in clean 1.5 ml Eppendorf tubes and the phages were precipitated again by addition of 200 µl (1/5 of the volume) 20% PEG8000, NaCl 2.5 mM to each tube. Following incubation on ice (30 min) and centrifugation (5 min, 14000 rpm, 4°C), the pellet was resuspended in 1 ml PBS supplemented with 15% glycerol and stored at -80°C.

Selection on immobilized protein H1X with previous depletion on TRX

The process of selection is the actual step of phage display. The phages express the nanobody on the so-called head of the phage, which allows the binding, i.e. a selection on a protein of interest. A previous depletion step can lower the number of phages used for the actual selection and reduces unspecific binding, i.e. not on the target, thus resulting in more positive hits in subsequent screening.

The selection process should ideally be started in the morning with inoculation of a 20 ml culture E. coli TG1TR in 2YT minimal medium and with incubation for approximately 6 to 8 h (37°C, 250 rpm) until an OD600 between 0.4 and 0.6.

One 1 ml aliquot of the frozen phages was thawed on ice and supplemented with 200 µl 20% PEG8000, 2.5 mM NaCl to precipitate the phages (30 min, ice) and remove the cryo-preservative glycerol. The phages were spun (5 min, 14000 rpm, 4°C) and the pellet was resuspended in 1 ml bovine serum albumin (BSA) (2% in PBS) and incubated in an overhead rotator (1 h, 4°C) for blocking. The immobilized TRX and H1X on magnetic beads (M450 Epoxy beads) were blocked in BSA (2%) as well. For depletion on TRX the blocking solution was removed from the beads and the resuspended phages were incubated with the immobilized proteins (2 h, overhead, 4°C). A 5 µl aliquot was recovered before incubation on TRX for later determination of the selection efficiency and referred as "input". After incubation, the depleted phages, i.e.

the supernatant of the TRX slurry, were incubated (2 h, overhead, 4°C) on immobilized H1X for selection. For elution of the bound phages, the beads were washed 9x with

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with 500 µl trypsin (1 mg/ml) (30 min, overhead, RT), 500 µl PBS were added and the supernatant was kept and referred as "output". An aliquot of 5 µl was stored for subsequent determination of the selection efficiency. The remaining 995 µl output were diluted with 4 ml 2YT medium and used for infection of 5 ml of the E. coli TG1TR

culture (0.4 < OD600 < 0.6) inoculated in the morning with subsequent incubation without agitation (30 min, 37°C). The infected bacteria were spun (10 min, 4000 rpm, RT) and the pellet was resuspended in 3 ml 2YT medium. 500 µl of the infected bacterial culture were plated out on square plates with 2YT-Agar supplemented with ampicillin (100 µg/ml) and glucose (2%) and incubated overnight at 30°C. The bacteria were harvested with 2x 3 ml 2YT medium containing ampicillin (100 µg) and glucose (2%). The bacterial suspension was spun (10 min, 4000 rpm, 4°C) and the pellet was resuspended in the same volume 2YT medium containing ampicillin and glucose as the pellet resulted an OD600 of approx. 100. Glycerol was added to a final concentration of 15% (v/v) and aliquots of 1 ml were stored at -80°C and used for the next round of phage display.

Determination of selection efficiency

For determination of the selection efficiency, the 5 µl input and output aliquots were diluted (1:100) in 2YT medium and titrated in 10x dilution steps until 10^-10 and 10^-7 for input and output, respectively. The diluted phages were used for infection of the same volume of the previously inoculated E. coli TG1TR culture (0.4 < OD600 < 0.6). The bacteria were incubated without agitation (30 min, 37°C) for infection. 100 µl of the infected bacteria were plated out on 2YT-Agar supplemented with ampicillin (100 µg/ml) and glucose (2%) and incubated overnight at 37°C. The following day, the colonies were counted to determine the selection efficiency.

Conservation of the selected clones

Two 96 deep well plates were prepared with 450 µl 2YT medium containing ampicillin (100 µg/ml) and glucose (2%) per well. 186 colonies of the output cultures (3 sterile controls) were picked and incubated overnight (37°C, 250 rpm). The next day, 150 µl of the bacteria cultures were transferred to round bottom 96 well plates and 30 µl glycerol (80%) were administered per well as cryo-conservative before storing the plates at -80°C.

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