V2R expression and purification

V2R Purification detailed protocol:

Buffers. (add fresh)

protocol

       Extraction and solubilization

1. Thaw cell pellets and add to lysis buffer 1L for 12L of culture
2. When lysis suspension is homogenous, centrifuge 15min at 38,400g @ 4ºC to pellet lysed cells.
3. Eliminate supernatant, suspend pellets in small amount (~10mL) of solubilization buffer. Solubilize (in 3 times with 100ml glass dounce tissue grinder with 15 and 20 strokes using A and B pestles) and pool samples. Add remaining solubilization buffer. Then add 1 mg/mL iodoacetamide.
4. Stir for 1h @ 4ºC, then add an additional 1 mg/mL iodoacetamide.

Strep-tag purification

1. During stiring, equilibrate resin (Strep-Tactin® Sepharose® resin) (10 ml) in solubilization buffer and pour it in four 500ml centrifuge tubes
2. Centrifuge Solubilization 15min at 38,400g @ 4ºC
3. Take out the supernatant and load it onto the Strep-Tactin resin
4. Incubate for 2h at 4 °C on an orbital shaker (170 rpm)
5. Centrifuge at 5,000g for 20min; Gently remove supernatant; Gently resuspend resin into 10ml wash buffer strep and transfer into 2 falcon tubes
6. Repeat two times: Centrifuge 5,000g for 8-10min; Gently remove supernatant; Gently resuspend resin into 30ml, last time pour into a gravity column.
7. Wash and monitor with OD measurement (280 nm). Stop when it is clean.
8. Elute in 2.5mM desthiobiotin-supplemented buffer

Flag-tag purification (equilibrate column with wash strep buffer)

1. Supplement eluate with 2 mM CaCl₂ and load onto a M1 anti-Flag affinity (gravity column) (Sigma-Aldrich) (12ml)
2. Wash in buffer 1 10 cv and buffer2 10cv
3. Elute

Size exclusion chromatography

1. Concentrate to 450μl with 50-kDa molecular weight cutoff (MWCO) concentrator (Millipore)
2. Inject on Superdex 200 increase (10/300 column)
3. Pool the fractions of interest and concentrate