III. Procedures
III.2. Preparations of the Proteins LC3 Wild Type, T6D, T12D, and ULD 26
III.2.1. Expressions of the Fusion Proteins Wild Type, T6D, and T12D
1. Transformed 100 μl competent cells BL21-CodonPlus-RIL with 100-160 ng the expression vector of the fusion proteins, and others followed the procedures just described (steps 8-9, p. 24). Note: the plates could be kept at 4°C for one week.
2. Inoculated 5-ml LB medium containing 50 μg ml−1 carbenicillin and 35 μg ml−1 chloramphenicol with a single colony selected from the plate, grown to saturation overnight at 37°C with shaking. Note that a 5-ml saturated culture is suitable for the efficient propagation in 1 liter medium during the expression; if more than 1 liter of the cell culture for protein expression were wanted, more 5-ml small cultures should be prepared (e.g., two 5-ml small cultures to expand into two 1-L cultures).
3. Inoculated each liter of the Carb+ & Chlor+ LB broth in a 2000-ml cell culture flask with each 5-ml of the saturated culture.
4. Grew the cells at 37°C with shaking at 250 r.p.m. to OD600 approximately 0.5 (Note:
this usually took about 5 hr), added 100 mg IPTG (the final concentration 0.42 mM), lowered the temperature to 22°C, and continued shaking overnight. Note: lowering the temperature may improve the solubility of the MBP-fusion protein inside the cells.
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III.2.2. Purifications of the Fusion Proteins MBP-LC3BG120 Wild Type, T6D, T12D, and ULD
1. Harvested the cells by centrifugation for 30 min at 12,000 g, 4°C; resuspended the cell pellet with 15-35 ml the amylose resin column buffer. Note: the cell resuspension could be snap frozen with liquid nitrogen, and stored at -20 °C for months.
2. Added 1 mM PMSF (from 500x stock, 500 mM) and trace DNase I before cell lysis;
lysed the cell in the resuspension on ice by the sonicator for 1 min, repeating 4 times, with 1-min cooling between each repeat; then centrifuged the disrupted cells for at least 30 min at 5,000 g, 4°C.
3. Applied the supernatant from 1-L cell culture to a column containing the amylose resin equilibrated with the column buffer. Note: 3 ml resin is sufficient to capture the tagged protein of the cell lysate from 1-L culture, and the volume of the resin used could be scaled up with the volume of the cell cultured (e.g., 6 ml resin to capture the proteins from 2-L cell culture).
4. Washed the column with 20-35 ml the column buffer; then eluted the bound fusion protein (e.g., MBP-LC3) with 4 successive 3.5-10 ml deionized water washes. Note:
the volumes of the column buffer for wash depended on the protein variants, and they could also be scaled up with the volumes of the cell lysate.
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Caution: for good reuse of the amylose resin, the procedure to regenerate (to clean)
it should be taken immediately after the chromatography (see p. 97 in Appendices for the procedure).
5. Identified the fractions containing the fusion protein by SDS-PAGE (see Figs 7-10).
Note in this study, the typical condition for SDS-PAGE was running a 15% gel with constant 120 V, 90 min.
6. Pooled the fractions containing the fusion protein, dialyzed and concentrated the sample approximately twofold with the Factor Xa reaction buffer, using the Amicon Ultra-15, 10 kDa by centrifugation at 5,000 g, 4°C.
III.2.3. MBP Removal from the Purified Fusion Proteins
1. Added the Factor Xa protease, 0.27 U per nmole of the substrate (i.e., the fusion protein) at 4°C. Note: the lower temperature could reduce non-specific substrate cleavages. Caution: the time appropriate for this reaction depended on the protein variants: it took one week for LC3 wild type and T6D, but for LC3 T12D it should be less than 3 days otherwise the unwanted protein fragments from over-digestion could significantly appear; by contrast, for MBP-ULD the reaction could be
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undergone at room temperature for 3 days while most of the ULD seemed to remain intact (based on the MS result, Fig. 24) although many unknown bands were seen on the SDS-PAGE gel (Fig. 14).
2. Dialyzed the digest again with 5 L the amylose resin column buffer using the SnakeSkin tube 7K MWCO overnight at 4°C.
3. Applied the digest to the amylose resin column equilibrated with the column buffer;
generally it took about 3 ml amylose resin for the effective capture of any MBPs (i.e., the free MBP & the remaining fusion protein) from 1-L cell culture.
4. Collected the flow-through containing the MBP-free LC3, and further eluted the residual free LC3 in column with 10-14 ml the column buffer. Note: for eluting the LC3 from more than one liter culture, additional repeats of the buffer wash could be done for “N-1” times (N: volume of cell culture in liters); afterward, the bound tag (MBP) or the remaining intact fusion protein might be washed out by 10 ml deionized water and checked with the following SDS-PAGE.
5. Ran SDS-PAGE to check the purity and the abundance of the obtained free LC3 (Figs 11-14). Note: if the LC3 eluted still showed visible MBP band on the gel, simply repeated the steps 3-5 using the regenerated amylose resin (Fig. 15).
6. Pooled the purified free LC3 for the further gel-filtration. Caution: the pool could be snap frozen with liquid nitrogen and kept at -20 °C for months, but not suitable
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for long-term storage in the liquid solution form due to the presence of the residual Factor Xa activity.
III.2.4. Further Purification of the LC3 with FPLC
Since the samples were prepared for CD spectropolarimetry, any impurities (e.g., trace tag MBP or other proteins from cell lysate) should be thoroughly removed through gel-filtration (i.e., fast protein liquid chromatography, FPLC) after the affinity chromatography.
1. Concentrated the LC3 through the affinity column with the Amicon Ultra-15, 7 (or 10) kDa by centrifugation at 5,000 g, 4°C. Note: typically, the free LC3 per liter of the cell culture could be finally concentrated to about 500 μl at this step, but this must be done at the lower temperature (i.e., 4°C) to avoid massive protein aggregation, and immediately followed by the gel-filtration to prevent the protein concentrate from the residual Factor Xa activity.
2. For injection, the concentrated samples needed to be high-speed centrifuged at 20,000 g, 5 min, and drew only the supernatant by syringe. Caution: the step 2 was recommended to perform at 4°C since the higher temperature (e.g., room temperature) could induce massive protein aggregation, which is not suitable for
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injection, and this was particularly the case when preparing the ULD.
3. Injected about 100 μl of the concentrated LC3 to a prepacked Superdex 75 (10 × 300-310 mm, bed volume approximately 24 ml) pre-equilibrated with the working buffer, and then collected effluent fractions (0.3 ml per fraction) at a flow rate 0.5-0.75 ml min-1 by the FPLC controller, AKTApurifier 10. Note: performed the whole gel-filtration at 4°C.
4. Pooled multiple fractions showing the right peak (known empirically, from the previous identifications by mass spectrometry) of high absorbance at 280 nm, as measured by the built-in spectrophotometer (around the 39th-46th fractions, see Figs 16-19). Note: the pooled, collected fractions could be kept at 4°C for a week or stored at -20°C for months.
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III.2.5. Protein Identity and Purity Check by MS
1. Concentrated the purified LC3 with Amicon Ultra-0.5 3 kDa by centrifugation to about 50 μM.
2. Drawing 50 μl of the protein concentrate for desalting using ZipTipC4: first acidified the sample with 5.6 μl 1% TFA, then following the manufacturer’s instruction using the solutions all described at p. 18 in the Reagent Setup.
3. Drying the desalted protein sample using the concentrator SpeedVac SPD111V: set at 35°C and spun 2 hr.
4. Sent the sample for molecular determination using G2 HDMS by the operator of MS Facility in Institute of Biological Chemistry, Academia Sinica.
Note: all protein molecular weights were estimated by their average molecular weights using the online calculator at http://www.expasy.org/; with the estimated values specified on the MS results (Figs 21-24).
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III.3. Observations of the LC3s Using CD Spectropolarimetry
III.3.1. Measurements of CD Spectra of the LC3s
1. Sample preparation: CD measurements was performed in a 1-mm cell, the volume of the 50 μM LC3 solution needed was 500 μl. For protein concentration determination, the absorbance at 280 nm was measured and corrected with the baseline extrapolated by the absorbance at 300 nm and 400 nm (as demonstrated in Fig. 25).
2. Equipment preparation. Caution: turned on the nitrogen, and flushed the optics compartment for at least 20 min before starting the machine for the nitrogen gas to displace the lens-damaging oxygen.30 Caution: turned on the lamp before turning on the computer, since electronic boards or computers could be destroyed by the voltage surge due to lamp firing if they were turned on prior to the lamp.31
3. Started the CD collection program: set the data path of the program for data storage;
the bandwidth 2.0 nm; the wavenlength range: from 250-200 nm; the wavelength interval (data pitch) 0.05 nm; the response time for 0.25 sec. Principle: most CD machines maintain constant current by raising the photomultiplier tube (PMT) voltage (on a Jasco it is called HT voltage) as the light intensity decreases, which is often the case when scanning at lower wavelengths; once the PMT voltage exceeds 500 V, the signal-to-noise ratio will greatly lower, rendering the data unreliable.31
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4. Cleaned the cell with distilled water by flushing for at least 5 times, filled with the protein sample and collected the CD spectrum at room temperature. Collected the data three times to make sure that the signals were not changing as a function of time, and then averaged the data sets. Saved the raw data on the hardware, and saved the data of each sample in separate text files with ellipticity values, [θ] (y-axis) as a function of wavelength (x-axis) so that it could be plotted to estimate protein conformations. Note: since the binary files can only be accessed by the corresponding CD machine and cannot be edited or imported into other editor (e.g., Microsoft Excel) or analysis program, the data in text format (.txt) must also be saved separately.31
III.3.2. Urea Denaturation Curves of the LC3 Wild Type, T6D, T12D, and ULD
1. Separate solutions were used to determine each point in the urea denaturation curves.
Experimental solutions were prepared volumetrically from replacing incremental amount of the original protein solution with the denaturant stock solution having the same volume. See Fig. 30 for the conceptual illustration and Table 3 for the practical setting.
2. Started the CD collection program, and entered Data Monitor mode: set the
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bandwidth 5.0 nm, the wavenlength 222 nm, the response time 16 sec, and at room temperature. Manually recorded the steady ellipticity values (solution in equilibrium) for each increase of the denaturant.
3. Plotted the ellipticity values (y-axis) against urea concentrations (x-axis) with Microsoft Excel.
III.3.3. GuHCl Denaturation Curves of the LC3 Wild Type, T12D, and ULD
1. Separate solutions were used to determine each point in the GuHCl denaturation curves. Experimental solutions were prepared volumetrically from replacing incremental amount of the original protein solution with the denaturant stock solution having the same volume as in measuring the urea denaturation curves. See Fig. 30 for the conceptual illustration (taking measuring the urea denaturation curve for example) and Table 4 for the practical setting.
2. Started the CD collection program, and entered Data Monitor mode: set the bandwidth 5.0 nm, the wavenlength 222 nm, the response time 16 sec, and at room temperature. Manually recorded the steady ellipticity values (solution in equilibrium) for each increase of the denaturant.
3. Plotted the ellipticity values (y-axis) against urea concentrations (x-axis) with Microsoft Excel.
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