Sequence-verified plasmids were transformed into NEB5a strain E. coli (New England Biolabs) (because the promoter in the pNCST vector is semi-constitutive in most strains of E. coli, we find it convenient to use a single strain for cloning and expression), plated on LB/agar supplemented with carbenicillin (100 μg/ml), and incubated overnight at 37°C. For proteins that matured efficiently at 37°C (AvicFP-F64L, mAvicFP1, AvicFP4, AausFP1, AausFP2, EGFP, mEGFP, and mNeonGreen), colonies were picked and inoculated directly into a 200-ml baffled wide-mouth flask containing 50 ml of 2xYT broth and 100 μg/ml carbenicillin, and incubated overnight at 37°C with shaking at 250 rpm. For proteins requiring multiple days at room temperature to mature (avGFP, AvicFP1, AvicFP2, AvicFP3, AausFP3, and AausFP4), a single colony was resuspended in 10 ml of 2xYT medium, and 100 μl of this suspension was plated on 5 100-mm petri dishes containing LB/agar and 100 μg/ml carbenicillin. After overnight incubation at 37°C to initially establish colonies, plates were then incubated at room temperature for several days in the dark.
Bacteria containing the recombinant protein were recovered by centrifuging liquid cultures in 50-ml conical tubes at 4,500g for 10 minutes. For proteins expressed on LB/agar plates, a razor blade was gently glided over the surface of the agar, harvesting the colonies on the blade, and then wiped into 2-ml microcentrifuge tubes and gently centrifuged to the bottom of the tube. Four milliliters of the lysis reagent B-PER (Thermo 78248) was added for every gram of E. coli pellet. Tubes were gently vortexed until the pellets were completely dissolved, taking care not to form bubbles from the detergent component of the B-PER. The resulting suspension was then incubated on a gentle rocker for 15 minutes and then centrifuged at >20,000g for 10 minutes to pellet insoluble debris. Note that we find that there is a strong correlation between true protein solubility and extraction efficiency in B-PER that is not true of other extraction methods such as sonication, which can solubilize aggregated FPs more readily.
Meanwhile, we prepared a purification column by adding 1–2 ml of Ni-NTA resin slurry (Expedeon) into a 15-ml gravity column (Bio-Rad), allowing the storage buffer to drip through. The column was equilibrated with 10 bed volumes of wash buffer (150 mM Tris [pH 7.5], 300 mM NaCl, 5 mM imidazole) and then capped at the bottom. After centrifugation, the lysate was directly added to the prepared Ni-NTA column. The column was then capped at the top and the lysate-resin slurry was tumbled end-over-end for 30 minutes at 4°C. The top/bottom caps were removed, and the liquid was allowed to drip through by gravity flow. The column was then washed 3 times with 3 column volumes of wash buffer. Finally, the protein was eluted from the column by gradual addition of elution buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 200 mM imidazole). Clear liquid was allowed to drip through, and only the fluorescent/colorful fraction was collected.
The proteins were then concentrated further using a 3-kD MWCO column (Amicon/Millipore) until the volume of protein solution was <150 μl. Meanwhile, 2× desalting columns (Pierce) were prepared for each protein by equilibrating in 50 mM Tris (pH 8.5)/150 mM NaCl according to the manufacturer’s instructions. Then 150 μl of protein solution was loaded onto the equilibrated desalting column and centrifuged at 1,500 rpm for 1 minute in a microcentrifuge. The collected protein was then passed through a second equilibrated desalting column to ensure complete buffer exchange.
Bacteria containing the recombinant protein were recovered by centrifuging liquid cultures in 50-ml conical tubes at 4,500g for 10 minutes. For proteins expressed on LB/agar plates, a razor blade was gently glided over the surface of the agar, harvesting the colonies on the blade, and then wiped into 2-ml microcentrifuge tubes and gently centrifuged to the bottom of the tube. Four milliliters of the lysis reagent B-PER (Thermo 78248) was added for every gram of E. coli pellet. Tubes were gently vortexed until the pellets were completely dissolved, taking care not to form bubbles from the detergent component of the B-PER. The resulting suspension was then incubated on a gentle rocker for 15 minutes and then centrifuged at >20,000g for 10 minutes to pellet insoluble debris. Note that we find that there is a strong correlation between true protein solubility and extraction efficiency in B-PER that is not true of other extraction methods such as sonication, which can solubilize aggregated FPs more readily.
Meanwhile, we prepared a purification column by adding 1–2 ml of Ni-NTA resin slurry (Expedeon) into a 15-ml gravity column (Bio-Rad), allowing the storage buffer to drip through. The column was equilibrated with 10 bed volumes of wash buffer (150 mM Tris [pH 7.5], 300 mM NaCl, 5 mM imidazole) and then capped at the bottom. After centrifugation, the lysate was directly added to the prepared Ni-NTA column. The column was then capped at the top and the lysate-resin slurry was tumbled end-over-end for 30 minutes at 4°C. The top/bottom caps were removed, and the liquid was allowed to drip through by gravity flow. The column was then washed 3 times with 3 column volumes of wash buffer. Finally, the protein was eluted from the column by gradual addition of elution buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 200 mM imidazole). Clear liquid was allowed to drip through, and only the fluorescent/colorful fraction was collected.
The proteins were then concentrated further using a 3-kD MWCO column (Amicon/Millipore) until the volume of protein solution was <150 μl. Meanwhile, 2× desalting columns (Pierce) were prepared for each protein by equilibrating in 50 mM Tris (pH 8.5)/150 mM NaCl according to the manufacturer’s instructions. Then 150 μl of protein solution was loaded onto the equilibrated desalting column and centrifuged at 1,500 rpm for 1 minute in a microcentrifuge. The collected protein was then passed through a second equilibrated desalting column to ensure complete buffer exchange.
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