Protein expression for crystal generation

Updated Expression and Purificaiton Protocol

Running/Lysis Buffer 1L keep refrigerated.

-          50 mM HEPES pH 8 (Tris pH 8 is fine too)

-         500 mM NaCl

-          0.5 mM TCEP 

-          20 mM Imidazole

-          Fill with DI H2O to 900 mL

-          Add 100 mL glycerol while stirring.

Elution Buffer 500 mL

-          50 mM HEPES pH 8 

-          1 M NaCl

-          500 mM TCEP

-          500 mM Imidazole

-          Fill with DI H2O to 450 mL

-          Add 50 mL glycerol (under sink) while stirring.

Resuspension

-          10 mL cold running buffer per gram cell paste.

-          1 EDTA-Free pic tab per 50 mL lysis (Pierce Cat#A32965).

-          Thoroughly resuspend on ice by pipetting.

Lysis with Sonication

-          Per 50 mL lysate, 5 s on/off cycles for 5 minutes of total sonicating at 80% power on ice. Sonicator used is a Fisherbrand Model 120 Sonic Dismembrator with a horn suitable for 50 mL sonication volumes.

Clarifying Lysate

-          Centrifuge at >18,000 xg for 30 minutes at 4^oC using the fixed angle rotor. Lysate should be clear and yellow.

-          Transfer to a new tube to separate soluble and insoluble fractions, make sure that no insoluble debris transfers over.

Purification

-             Purification was performed using a BioRad NGC Quest FPLC with a sample pump.

-             We use a BioRad Nuvia 5 mL IMAC column for affinity purification.  FPLC should have the running/lysis buffer loaded on pump A and the elution buffer on pump B.

-          Clean the column by flushing 3 CV of 100% pump B.

-          Equilibrate the column with 5 CV of pump A or until UV baseline is reached.

-          Place sample pump line into the clarified lysate and pull 5 mL through the pump to prime it and purge any air.

-         Load the sample directly onto the column, wash with 100 pump A until baseline is reached, do a linear gradient elution from 0 to 100% pump B.

-          Pick peak fractions and run 20 uL (with 20 uL load dye) on a SDS-PAGE gel along with ladder.

-          Combine fractions with the correct band corresponding to protein of interest take a reading on the nanodrop. A260/280 should be <0.8. If greater, add DNAse. Use extinction coefficient to determine molarity.

TEV Cleavage and Dialysis

-          Add 1:5,000 HisTev, place in dialysis membrane, and dialyze against 4 L of running buffer overnight at 4^oC with stirring.

Reverse Ni-NTA

-          Load dialyzed protein onto the IMAC column as before, collect flow through. Run 20 mL running buffer over column and collect. In a separate fraction, run 100% pump B/elution buffer to elute the cleaved His tag and His-TEV.

-          While this is running make SEC buffer, which is running buffer without imidazole. Degas for at least 30 minutes with stirring. Once reverse Ni-NTA protocol is complete, start equilibrating the SEC column at 1 mL/min for at least 1 CV.

Gel Filtration

-          Using a spin concentrator with a 30 kDa molecular weight cutoff, concentrate the protein to at least 5 mg/mL using the filtration concentrators. Use the swinging bucket rotor at 4^oC, 4,000xg for 5-15 minutes at a time. Concentrated to no less than 1 mL, ideally to 5 mL. Protein should not be concentrated to more than 20 mg/mL, make sure you are taking nanodrop readings throughout the concentrating. Ideally, the A260/280 ratio will decrease as the A280 increases. Remember that the absorbance isn’t always 1:1 with the A280 given the extinction coefficient of the protein, but in general you need an A280 of at least 3 to see fractions on the SEC.

-          Run fractions on the gel. ER LBD typically elutes around 30-60 kDa on the SEC. Concentrate to 5-20 mg/mL and flash freeze for later use. If time, set up crystal trays right away.

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