TITLE: Expression and Purification of Thermostable Proteins Expressed in E. coli
AUTHORS: Michael Van Dyke

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Our laboratory studies putative transcription regulatory proteins from the extreme thermophile, </span><span style = "font-style:italic;">Thermus thermophilus</span><span> HB8. To do so, these proteins are often expressed in the mesothermic organism </span><span style = "font-style:italic;">Escherichia coli</span><span> and purified from whole-cell extracts. Here we describe our standard expression and purification scheme and its analysis using the thermostable protein His6-GFP-TEV. His6-GFP-TEV is usually used as an N-terminus tag for the bacterial expression of recombinant proteins. However, this surrogate provides a facile visual indicator of thermostable protein expression and purification and is especially amenable for laboratory instruction in a primarily undergraduate educational environment.</span></div></div>

[STEPS]
?. [Protein expression]
Inoculate 1 mL LB:50 µg/mL kanamycin culture in a 5 mL Falcon snap-cap tube with a ~1 cm swatch from a streak. Repeat for a second clone. Incubate tubes in a rack at 37 °C, 240 rpm, 30 min. Cultures will appear slightly cloudy when well dissociated.
?. [Protein expression]
Use each 1 mL culture to inoculate a 50 mL SOB:50 µg/mL kanamycin culture in a 250 mL flat-bottom flask. Culture should be relatively clear upon dilution. Incubate 37 °C, 220 rpm, for 2h.
?. [Protein expression]
Cultures should become slightly cloudy. Remove 1 mL aliquot and measure absorbance at 600 nm (A600). Values are typically 0.2-0.25 at this point. Continue incubation for an additional 30 min.
?. [Protein expression]
Remove 1 mL aliquot and measure absorbance. A600 should be between 0.4-0.5. Add 500 µL 100 mM IPTG to culture and continue incubation at 37 °C, 220 rpm, for 4h. Process aliquot by transferring to 1.5 mL tube, microcentrifuging 1 min, decanting medium, resuspending with 20 µL 2x BEB (40 mM Tris-Cl [pH 7.5], 200 mM NaCl, 0.2 mM EDTA, 2 mM DTT, and 1 mM PMSF), and freezing pellet at –20 °C. The tube should be labeled with protein name, clone number, and time (e.g., “NGFP G1 1330h). This is also referred to as the logarithmic growth (“log”) sample.
?. [Protein expression]
Cultures should have a slight yellow-green hue after 4h induction. Remove 1 mL aliquot and measure A600 (typically 1.8-1.9). Process aliquot as described in Step 4 above. This is also referred to as the IPTG-induced (“induced”) sample.
?. [Protein expression]
Transfer remaining culture equally into two 50 mL conical screw-cap polypropylene centrifuge tubes. Centrifuge tubes at 4000 rpm for 10 min at 4 °C. Decant supernatant carefully, as pellets may be loose. Resuspend each pellet with 500 µL 2x BEB by mild vortexing. Pool and quick freeze (liquid N2) before storage at –20 °C.
?. [Protein purification]
Thaw frozen pellets on ice. Measure volume (in this example, 1250 µL) and transfer to a 2 mL microcentrifuge tube. Save 100 µL aliquot of freeze/thawed sample (“F/T”). To the remainder, add 0.02x volume (23 µL) 10 µg/µL freshly prepared lysozyme and mix by inversion. Incubate 10 min on ice, mixing every 5 min.
?. [Protein purification]
Using a microprobe, sonicate at minimum power (2.5) for 10 sec., then chill on ice for 50 sec. Repeat a total of five times. Note that clarification of the suspension is apparent after the third burst. If sudsing occurs, microfuge briefly (4 sec) to reduce before sonicating again.
?. [Protein purification]
Microcentrifuge samples for 10 min at 4 °C. Transfer supernatant to a 1.5 mL microcentrifuge tube. Measure volume (1100 µL). The supernatant should be clear and intensely yellow-green; pellet, relatively small (20 µL) and slightly green, indicating efficient bacterial lysis and release of soluble NGFP. Save 100 µL aliquot of lysis/soluble sample (“L/S”).
?. [Protein purification]
Place samples into a 70 °C hot block. Remember to add a small amount of water to each well to facilitate good heat transfer. Incubate 15 min with samples mixed by inversion after 8 min. Samples will become cloudy due to the denaturation of E. coli proteins. An intense yellow-green color should be retained.
?. [Protein purification]
Microcentrifuge samples for 15 min at 4 °C. Transfer supernatant to a 1.5 mL microcentrifuge tube. Measure volume (900 µL). The supernatant should be intensely yellow-green; pellet, larger (100 µL) and beige in color, indicating efficient E. coli protein denaturation and soluble NGFP protein recovery. Save 100 µL aliquot of the heat-treated sample (“Δ”).
?. [Protein purification]
NGFP can be stored frozen at –20 °C. To avoid repeated freeze/thaw cycles, NGFP can be diluted with an equal volume of glycerol and mixed by inversion, then gentle rocking for 30 min at 4 °C before storage at –20 °C. A 1x BEB:50% glycerol solution will not freeze under these conditions.
?. [Protein analysis: Fluorescence]
SampleQubit Fluorescence2xBEB (blank)29.321330h (log)64.031800h (induced)109729.28F/T131246.60L/S147702.10Δ143622.60
SampleQubit Fluorescence2xBEB (blank)29.321330h (log)64.031800h (induced)109729.28F/T131246.60L/S147702.10Δ143622.60
?. [Protein analysis: SDS-PAGE]
Our laboratory routinely performs SDS-PAGE analyses using homemade 12% Mini-Protean SDS-PAGE gels and associated reagents. Recipes and instructions may be found in the Bio-Rad Bulletin_6040 (https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf).
?. [Protein analysis: Fluorescence]
NGFP is a highly fluorescent protein. Thus, fluorescence can be used to measure recovery at each step during purification.
?. [Protein analysis: Fluorescence]
Dilute 1 µL each sample with 200 µL 2x BEB in a 0.45 mL Corning Axygen super-clear microfuge tube. The control contains only 2x BEB. Remember to label only the tube caps.
?. [Protein analysis: Fluorescence]
Fluorescence assays using an Invitrogen Qubit 3 fluorometer:1. Turn on the Qubit 3 fluorometer. 2. From Home Screen, choose “Fluorometer” mode.3. Next, choose Blue [470 nm] excitation.4. Insert sample and close lid.5. Press “Read tube.”6. Record green emission [510 – 580 nm] value shown.7. Remove the tube and insert a new sample. Repeat steps 15.4 – 15.7.
?. [Protein analysis: SDS-PAGE]
Dilute 2 µL each sample with 20 µL 1x Laemmli sample buffer + BME in a 0.45 mL microfuge tube. Heat sample at 95 °C for 10 min (unless otherwise noted), minispin briefly to consolidate sample, then keep at room temperature until needed.
?. [Protein analysis: SDS-PAGE]
SDS-PAGE analysis using a Bio-Rad Mini-Protean gel system:1. Assemble gel in electrophoresis cell. 2. Fill inner and outer chambers with 1x Tris-glycine/SDS running buffer. 3. Slowly, load 20 µL of the sample into gel well. Samples containing whole bacteria (e.g., “log” and “induced”) may exhibit properties (viscosity, stringiness) that make them more difficult. Repeated pipetting before loading may be beneficial.4. Electrophorese at 50 V for 20 min, then 200 V until bromophenol blue indicator dye band is near the bottom of the gel (60 min total). 5. Disassemble electrophoresis cell, then gel. 6. To image native NGFP directly, we use a GelDoc EZ Imaging System, Blue tray, and the fluorescein option. Details may be found at https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10019683.pdf. 7. Afterward, the gel is stained with Coomassie Brilliant Blue R250 and destained until the background is clear. Imaging is performed with a GelDoc EZ Imaging System, White light tray, and the Coomassie option.
?. [Protein analysis: SDS-PAGE]