Biotinylation of Membrane Proteins for Binder Selections

Design FX-cloning compatible primers targeting your gene of interest. This is most conveniently done online at the https://www.fxcloning.org https://www.fxcloning.org website using a FASTA-formatted sequence including a start and stop codon. Order the primer set optimized toward removal of stable hairpin structures ( see Note 1 ).

Amplify the gene of interest by PCR. Prepare a 50µL and add the DNA polymerase immediately prior to starting the reaction.

Use a touch-down [21] program, e.g., (1) 0h 0m 30s at 98°C;

(2) 0h 0m 10s at 98°C;

(3) 0h 0m 15s at 61°C (annealing temperature decreased by 0.5°C per cycle);

(4) 15- 30 s/kb at 72°C; repeat (2)–(4) 14 times;

(5) 0h 0m 10s at 98°C;

(6) 0h 0m 15s at 53°C;

(7) 15–30 s/kb at 72°C; repeat (5)–(7) 14 times;

(8) 0h 2m 0s at 72°C;

(9) unlimited at 10°C.

Analyze the product on a preparative TAE agarose gel. Purify the relevant band using a DNA gel extraction kit and quantify the DNA spectrophotometrically.

Mix 50ng with the extracted insert in a molar ratio of vector:insert of 1:5 ( see Note 2 ).

Add 1µL and adjust the volume to 9µL with ultrapure water. Add 1µL and incubate for 1h 0m 0s at 37°C in a PCR machine.

Heat inactivate the SapI for 0h 20m 0s at 65°C. Let the sample cool down and add 1.25µL and 1.25µL. Incubate for 1h 0m 0s at 65Room temperature.

Heat inactivate the T4 DNA ligase for 0h 20m 0s at 65°C and transform 5µL to 100µL.

Plate 10% and 90% aliquots on LB-agar-Cam plates and incubate 0h 20m 0s at 37°C.

Use a single colony to inoculate 5mL and cultivate 0h 20m 0s. Isolate the plasmid using a miniprep kit, determine the concentration spectrophotometrically, and verify the insert by DNA sequencing using the pINIT_cat sequencing primers.

Mix 50ng ( see Note 3 ) with pINIT_cat carrying the insert to a molar ratio of vector:pINIT_cat-derivative of 1:5. Add 1µL and adjust the volume to 9µL with ultrapure water. Add 1µL and incubate for 1h 0m 0s at 37°C in a PCR machine.

Heat inactivate the SapI for 0h 20m 0s at 65°C. Let the sample cool down and add 1.25µL and 1.25µL. Incubate for 1h 0m 0s at 65Room temperature.

Heat inactivate the T4 DNA ligase for 0h 20m 0s at 65°C and transform 5µL to 100µL.

Plate 10% and 90% aliquots on LB-agar-Amp plates. Incubate the plates 0h 20m 0s at 37°C.

Use a single colony to inoculate 5mL and cultivate 0h 20m 0s at 37°C.

Archive the culture as a glycerol stock at -80°C ( see Note 4 ). This stock can serve for inoculation of expression cultures based on the araBAD promoter ( see Notes 5 & 6 ).

Recombinantly express the target protein using previously established procedures [22] ( see Note 7 ). Purify the Avi-tagged target protein ( see Note 8 )and determine the protein concentration spectrophotometrically.

Add 3C protease to a molar ratio of 1:10 to cleave off the decaHis-tag while dialyzing the sample for 1h 0m 0s at 4°C to remove excess imidazole ( see Note 9 ).

Adjust the target protein concentration to 10micromolar (µM) – 50micromolar (µM) (either by dilution or using a concentrator unit). Add biotin to a molar ratio of target protein:biotin of 1:1.5, 5 mM ATP, 10 mM MgOAc and BirA to a molar ratio of target protein:BirA of 20:1 ( see Note 10 ). Incubate the sample 1h 0m 0s at 4°C ( see Note 11 ).

Remove His-tagged BirA, HRV 3C protease, and potential remaining contaminants from the sample by reverse IMAC and collect the flow-through holding the biotinylated target protein.

Perform size exclusion chromatography (SEC) to remove soluble aggregates and excess of biotin from the sample ( see Note 12 ). Determine the degree of biotinylation as outlined in Section 3.5.

Proceed with the selection of binders such as nanobodies and sybodies ( see Note 13 ) or store the target protein ( see Notes 14 & 15 ).

Generate mammalian expression vectors for the gene of interest in pC031 or pC039 to obtain a fusion protein with an N- or C-terminal Avi-tag ( see Notes 16 & 17 ).

Split an Expi293 subculture ( see Note 18 ), typically grown to 3-5 × 10⁶ cells/mL, into a 3 L Fernbach shaking flask and adjust to a final volume of 0.6L with a density of 0.7 × 10⁶ cells/mL.

Incubate the culture for 72h 0m 0s at 37°C, under humidified atmosphere and 5% CO₂ in a shaking incubator.

On the day of the transient transfection, adjust the culture to 830mL with a density of 3.4 × 10⁶ cells/mL by adding Expi293 medium and/or removing cells.

Add 20mL (final concentration of 50micromolar (µM)) ( see Note 19 ).

Pipet 50mL into a 100 mL sterile Schott bottle. Add 2.7mL, shake gently, and incubate for 0h 5m 0s at 37Room temperature.

Pipet 50mL into a second 100 mL Schott bottle and add the two plasmid batches in a final amount of 1mg to 0.1mg, target protein expression plasmid:BirA expression plasmid, respectively. Shake gently and incubate for 0h 5m 0s at 37Room temperature.

Mix the contents of both bottles, filter sterilize, and incubate for 0h 20m 0s – 0h 30m 0s at 37Room temperature to form the transfection complex.

Add 100mL to the Fernbach shaking flask with 850mL for a final volume of 950mL. Incubate further at 37°C and 5% CO₂ with mild shaking.

Add sterile 5mL and 50mL from the ExpiFectamine 293 transfection kit at 16h 0m 0s – 20h 0m 0s post-transfection and continue incubation.

Incubate for a total time of approximately 48h 0m 0s - 72h 0m 0s post-transfection depending on the most optimal condition for protein expression. Harvest the cells by centrifugation at 3000x g, flash freeze the pellet in liquid nitrogen, and store at -80°C.

Purify the biotinylated Avi-tagged target protein ( see Note 8 ) and determine the protein concentration spectrophotometrically. Determine the degree of biotinylation as outlined in Section 3.5. Proceed with the selection of binders such as nanobodies and sybodies ( see Note 13 ) or store the target protein ( see Notes 14 & 15 ).

Recombinantly express the target protein using previously established procedures [22]. Purify the target protein and employ preparative SEC using PBS, supplemented with the required detergent, as buffer ( see Note 20 ). Determine the protein concentration spectrophotometrically.

Concentrate the target protein to 50micromolar (µM) - 200micromolar (µM).

Dissolve EZ-Link Sulfo-NHS-LC-Biotin in highly pure DMSO to a concentration of 10millimolar (mM) ( see Note 21 ).

Add EZ-Link Sulfo-NHS-LC-Biotin to the target protein in fivefold molar excess and incubate at 25°C for 0h 30m 0s under gentle agitation ( see Note 22 ).

Perform SEC to remove excess of biotin from the sample ( see Note 12 ).

Determine the biotinylation pattern of the biotinylated target protein by mass-spectrometry (Fig. 1, see Note 23 )). In case mass spectrometry analysis is not available or cannot be carried out due to the target’s high molecular weight, determine the degree of biotinylation as outlined in Section 3.5.

Proceed with the selection of binders such as nanobodies and sybodies ( see Note 13 ) or store the target protein ( see Notes 14 & 15 ).

Mix two aliquots of 10µg with 5× SDS-PAGE sample buffer.

Add streptavidin to one of the aliquots in a 1:1 molar ratio of target protein:streptavidin ( see Note 24 ).

Analyze the control (no addition) and test (streptavidin addition) samples in adjacent lanes on SDS-PAGE.

Stain the gel with Coomassie Brilliant Blue R-250 and quantify the band intensities with the ImageJ software and calculate the degree of biotinylation ( see Notes 25 & 26 ).