Identification of interacting proteins using PUP-IT

Protein proximity labeling has been developed to identify protein-protein interactions. Here we report a tagging method termed PUP-IT (pupylation based interaction tagging) where a bacterial PUP ligase is fused to the bait protein and this chimeric protein mediates the covalent modification of prey protein with Pup protein. Pup is a small protein containing 64 amino acids. The N terminus of Pup can be fused to the bacteria-derived biotinylated BCCP domain. Therefore, any protein that modified by Pup can be enriched by streptavidin pull down under denaturing condition, which is often used by other types of sample preparation for mass spectrometry identification. Here we describe two ways to achieve Pup labeling in cells. One is based on transient transfection and another based on the inducible cell line. After labeling in cells, the Pup labeled target proteins can be enriched by using the same streptavidin pull-down procedure.

(2) The day before transfection, harvest Jurkat cells by spinning at 800 g for 5 min at room temperature, then re-suspend in pre-warmed RPMI 1640 (with 10% FBS, no Penicillin-Streptomycin) at 0.4 million/ml.
(4) Harvest cells by spinning at 800 g for 5 min at room temperature.
(9) After the pulse, allow cells to recover in cuvette for 15 min at room temperature in the cell culture hood.
(10) Gently transfer cells to pre-warmed recovery media and maintain them in cell culture incubator.
(12) Between 36~48 hours after transfection, check cell transfection efficiency by flow cytometer. The GFP positive cells are usually between 65-80% in living cells.
(13) Harvest cells by spinning at 800 g for 5 min at room temperature, then wash cells with 1 ml cold PBS for three times. Cells can be immediately lysed for sample preparation or stored in -80 °C until use.
(5) FACS sort BFP positive cells into 96 well plates with 1 cell/well for single clone selection.
(6) Three weeks after sorting, examine BFP expression in each clone by flow cytometer with or without doxycycline induction (2 μg/ml).
(2) For each sample, 10 7 cells (typically 20 ml resuspended cells at 0.5 million/ml) are aliquot to each 75 cm2 cell culture flask. Add 20 μl 1000× doxycycline (final concentration 2 μg/ml) to each sample. (Optional: Biotin can also been added to media at 4 μM to ensure the consistency of labeling in different cell culture media).
(3) About 24 hours after induction, harvest cells by spinning at 800 g for 5 min at room temperature, then wash cells once with 1ml cold PBS. Cells can be immediately lysed for sample preparation or stored in -80 °C until use.

Part 2. Streptavidin Pull Down of Bio-PUP(E) Modified Proteins
Day 1: (1) Lyse 10~30 million cells in 1 ml lysis buffer supplemented with protease inhibitor cocktail on ice for one hour.
(2) Clarify the lysates by centrifuging at 13000 rpm for 10 min at 4 °C.
(3) Transfer 900 μl cell lysate to a new 1.5 ml centrifuge tube, add 576 mg urea powder to cell lysate (final volume is around 1.2 ml and the urea concentration is around 8 M). The dissolving of urea absorbs heat and rotating the tube on a rotater at room temperature can accelerate this step. *NOTE: Take 10 μl cell lysate for western blot with streptavidin-HRP and anti-Myc blotting to confirm the expression and activity of PUP-IT.
(4) Add 12 μl of 1 M DTT (final concentration 10 mM) to cell lysate, shake at 600 rpm at 56°C for 1 hour. *NOTE: The purpose of steps 4~6 is to reduce and alkylate cysteine residues. These steps are optional.
(7) Add 50 μl streptavidin magnetic beads to each sample and incubate on a rotator at 4°C overnight.

Day 2:
(1) Place sample tubes on a magnetic stand, wait till all the brown magnetic beads settle on the side of the centrifuge tube (about 1~2 minutes) and aspirate out lysate.
(2) Add 1 ml Wash Buffer 1 to each tube and shake at room temperature for 5 min on eppendorf mixer at 1000 rpm.
(3) Place sample tubes on magnetic stand and aspirate out Wash Buffer 1.
(5) Wash beads once with Wash Buffer 2. Add 1 ml Wash Buffer 2 to each tube and shake at room temperature for 5 min on eppendorf mixer at 1000 rpm. Place tubes on magnetic stand and aspirate out Wash Buffer 2.
(6) Wash beads with Wash Buffer 3. Add 1 ml Wash Buffer 3 to each tube and shake at room temperature for 5 min on eppendorf mixer at 1000 rpm. Place tubes on magnetic stand and aspirate out Wash Buffer 3.
(8) Wash beads once with Wash Buffer 4, then wash beads twice with Wash Buffer 5.
(9) Trypsin digest on beads. For each sample, dilute 6 μg trypsin in 125 μl buffer 5, add the diluted trypsin to the beads. Incubate samples at 37 °C overnight.

Day 3:
(1) Transfer the supernatant to a new tube, wash beads twice with 30 μl Washing Buffer 5.
Combine the supernatant with the washings.
(2) Concentrate the samples using SpeedVac concentrator to reduce sample volume to 8 less than 40 μl.
(5) Dry samples in the SpeedVac concentrator. The samples can be stored in -80 °C and are ready to be dissolved for mass spectrometry analysis.
A proximity-tagging system to identify membrane proteinprotein interactions by Qiang Liu, Jun Zheng, Weiping Sun, +5