Production of sensors in E. coli and protein purification

Protocol for protein purification (FLIP sensors) using His-Bind resin


Transform BL21(DE3)Gold (Stratagene) E. coli cells with your plasmid (containing His-Tag and your binding protein)

Pick a single colony from a fresh plate and inoculate into 50-100mL LB containing the appropriate antibiotics (no IPTG) at room temperature. Preculture seems unnecessary, but preculture or culture at 37 °C seems to suppresses the leaky expression from the lac promoter. Grow cells while shaking at RT for 2-3 days in darkness (to prevent damage to the fluorophores).

Note: Cell suspension can be tested for expression of the protein in a fluorimeter (Fig. 1). Pellet 1 ml of culture, discard supernatant and resuspend in 1ml Tris-Cl buffer pH8.0. This suspension can be used directly for taking a spectrum.

Pellet the cells and discard the medium. Resuspend cells in 5mL of cold 20mM Tris-Cl buffer pH 8.0. Even if the cells show little fluorescence (light yellow) you will probably get enough protein for titration. Transfer into a 15mL Falcon tube.

Sonicate to lyze the cells (5-6 times 10-15 sec, 15 sec pause, try to make no foam, keep on ice so that the protein does not get warm), centrifuge for about 1 hour to get rid of all the cell debris. (The color of the lysate at this stage is a good indicator of the protein amount. It should be yellow, if the lysate is colorless there is probably little protein, cf Fig. 1; The inset in the graph shows purified protein)

Filter (0.45µm) the lysate into a fresh 15mL Falcon tube and add the equilibrated His-bind resin to it.

In the cold room: Allow the protein to bind at 4 °C for 1-2 hr with gentle shaking, until the resin turns yellow. Reload everything to the column and allow all the supernatant to pass. Wash the resin with 5mL of cold wash buffer twice.

Elute the protein with 500µL-2mL of elution buffer (yield several mg).

Store at 4 °C. Wait overnight before doing measurements, the proteins seem to need time to "recover" from the isolation procedure.

Note: For best results, analyze protein within 1 week of isolation, as the ratio change tends to decrease with longer storage. It is possible to freeze the protein in glycerol.

Regenerate column by washing twice with strip buffer and store resin in strip buffer.

Equilibration of resin:
Add 1 mL His-Bind resin to column.
Wash with 5 mL water.
Apply 2 mL 50 mM NiCl.
Wash with 5 mL 20mM Tris buffer pH 8.0. Keep the resin wet until it is added to the protein.

Wash Buffer:
20mM Tris-Cl pH 8.0
10mM imidazole

Elution Buffer:
20mM Tris-Cl pH 8.0
500mM Imidazole
(No NaCl as YFP is sensitive to Cl-)

Strip Buffer:
500mM NaCl, 100mM EDTA, 20mM Tris pH 7.9

Hisbind resin: We use Novagen, # 69670
BL21(DE3)Gold from Stratagene
Fluorimeter: we use Safire Tecan
96 well Microtiter plates with flat bottom wells ( we normally do not use black plates)