Last time you used the lactose-analogue IPTG to induce expression of inverse pericam in BL21(DE3) bacteria. Today you will isolate IPC from the bacteria, and you will begin characterizing your wild-type and mutant proteins.
We can take several measures to ensure that a high quantity of plasmid-encoded protein is produced by our bacteria, such as using a high-copy plasmid. However, the bacteria in which we grow the protein clearly need to produce other proteins merely to survive. The bacterial expression vector we are using (pRSET) contains six Histidine codons downstream of a bacterial promoter and in-frame with a start codon. Our resultant protein is therefore marked by the presence of these additional encoded residues, or His-tagged. Histidine has several interesting properties, notably its near-neutral pKa, and His-rich peptides are promiscuous binders, particularly to metals. (For example, histidine side chains help coordinate iron molecules in hemoglobin.)
Affinity separation process
Green represents Nickel, blue the (His-tagged) protein of interest, and orange the other proteins in the cell extract.
Today we will use a Nickel-agarose resin to separate our protein of interest from the other proteins present in the bacteria. The His-tagged protein will preferentially bind to the Nickel-coated beads, while proteins irrelevant to our purposes in Module 2 can be washed away. Finally, a high concentration of imidazole (which is the side chain of histidine) can be used to elute the His-tagged inverse pericam by competition. Due to the inherent fragility of IPC, we will add several components to our protein extraction and purification reagents: bovine serum albumin (BSA), which is a protein stabilizer, and a cocktail of protease inhibitors.
Prior to purifying our protein, we will lyse the bacteria and save the whole cell extracts to later run on a protein gel. This procedure is called SDS-PAGE, for sodium docecyl sulfate-polyacrylamide gel electrophoresis. SDS is an ionic surfactant (or detergent), which denatures the proteins and coats them with a negative charge. Since denatured proteins are linear, they will move through the gel at a speed inversely proportional to their molecular weight, just like DNA on agarose gels. (Non-denatured proteins run according to their molecular weight, shape, and charge.) As we did with DNA gels, we will run a reference ladder containing proteins of known molecular weight and amount. When running –IPTG and +IPTG samples side-by-side, you should see the emergence of a protein band at the expected molecular weight for inverse pericam, which may be very faint or non-existent in the control sample, but bright and thick in the induced sample. To visualize all the proteins released by the bacteria, you will stain the gels with Coomassie Brilliant Blue (actually, a variant called BioSafe Coomassie). This is a non-specific stain for all proteins. In a technique called Western Blotting, SDS-PAGE is combined with the use of antibodies to preferentially stain a single protein.
After purifying inverse pericam from your bacterial lysates, you will measure the protein concentration by the Bradford colorimetric assay, named after the scientist who first published it. The dye used in this assay is the same one you will use to stain your protein gels – Coomassie. In acidic solution, Coomassie normally has an absorbance peak at ~ 465 nm (blue light), but this peak is shifted to 595 nm (orange light) upon binding to protein. Protein binding occurs primarily via arginine, as well as other basic and aromatic residues, as described here. The concentration of protein present in a sample is thus proportional to the 595 nm absorbance peak, and its absolute value can be determined using a standard curve of reference protein. We do not have a sample of inverse pericam with a known quantity of protein, so today we will use BSA as a reference protein. Because the compositions of IPC and BSA with respect to arginine may vary, this assay will really only give the relative concentrations of your protein samples, and the absolute concentrations will have an associated error.
Part 1: Lysis of cells producing wild-type and mutant IPC
- You will be given a 3 mL aliquot of room temperature BPER (bacterial protein extraction reagent), which also contains 0.1% bovine serum albumin (BSA, a stabilizer), and a protease inhibitor cocktail to guard against protein degradation. When you are ready to begin, add 1:1000 of cold lysing enzyme mixture (obtained from teaching staff) to the BPER solution.
- Per cell pellet (6 total), add the appropriate volume of enzyme-containing BPER and resuspend by pipetting until the solution is relatively homogeneous.
- Resuspend -IPTG samples in 300 μL, and +IPTG samples in 600 μL - do you remember why?
- Pipet up and down to mix. (May take a good 15-20x.)
- Incubate the solutions (at room temperature) for 7 min.
- During this incubation, you may begin the resin preparation described in Part 3.
- Finally, spin for 3 min. at maximum speed and transfer supernatants to fresh tubes.
- While one partner completes Part 2, the other partner can begin/continue with the resin preparation in Part 3.
- Last time you measured the amount of cells in each of your samples. (If you ran cultures overnight, the teaching faculty measured the +IPTG samples for you and posted the results.) Look back at your measurements, and find the sample with the lowest cell concentration. Set aside 15 μL of this sample for PAGE analysis in an eppendorf.
- For your other five samples, you should take the amount of bacterial lysate corresponding to the same number of cells as the lowest concentration sample. For example, if the OD600 of your WT -IPTG sample was 0.05, and the OD600 of your WT +IPTG sample was 0.30, you would take 15 μL of the -IPTG, but only 2.5 μL of the +IPTG sample.
- Next, add enough water so the each sample has 15 μL of liquid in it. You might use the table below to guide your work.
- Finally, add 15 μL of 2X sample buffer to 15 μL of each of your diluted lysates. These will be stored frozen until next time.
| Sample Name
|| Sample Volume (μL)
|| Water Volume (μL)
| -IPTG WT
| +IPTG WT
| -IPTG _______
| +IPTG _______
| -IPTG _______
| +IPTG _______
Part 3: Protein purification
Part 3A: Nickel-agarose purification
You will process three samples (the three +IPTG extracts) according to the following procedure. You should either time your spins with another group, or balance your tubes with 3-way symmetry. Keep all buffers on ice when not in use. All spins should be performed at 1000 rcf for 1 min.
- The following buffers have been prepared for you:
- Charge Buffer (50 mM NiSO4)
- Binding Buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9)
- Wash Buffer (0.5 M NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9)
- Elute Buffer (0.5 M NaCl, 10 mM Tris-HCl, 1 M imidazole, pH 7.9)
- All but the Charge Buffer contain protease inhibitors to help keep your protein intact.
- Gently rock the agarose resin to fully resuspend it, then distribute 400 μL of slurry to each of three tubes. Or, thank your TA if he has already pre-aliquoted them for you!
- Label each tube as wild-type or mutant, then spin for 1 min. at low speed.
- Remove the 200 μL of supernatant from the resin and add it to your waste collection tube. The damp, semi-solid resin left behind should be ~ 200 μL “tall.”
- First you must rinse the resin. Add 400 μL of sterile DI water to each tube. Place on the nutator for 15-60 seconds to mix, or simply invert the tube several times. Flick the tube to complete resuspension of the resin if necessary.
- Spin for 1 min., then pipet off and discard the entire supernatant (400 μL).
- Repeat steps 5 and 6 for the following buffers
- a second wash with DI water
- 3 washes with Charge Buffer, 400 μL each time
- 2 washes with Binding Buffer, 400 μL each time
- Add your entire cell extract to the resin (~550-600 μL, however much is left). Be sure to add each sample to the appropriately labeled tube!
- Invert to mix the three samples as usual, then place on the nutator for 5 min. Spin and discard supernatants as before.
- Now you will again repeat steps 5 and 6, to wash away contaminants:
- 3 washes with Binding Buffer, 600 μL each time
- 2 washes with Wash Buffer, 600 μL each time
- Finally, you will collect your protein. Add 500 μL of Elute Buffer, resuspend and spin as usual. Do not throw away the supernatant! Instead, transfer it to a fresh eppendorf tube, labeled “pure IPC X#Z,” “pure IPC E67K/T79P/M124S,” or “pure IPC WT.”
- Do not throw away the resin yet either! Instead, add another 500 μL of Elute Buffer, and repeat the step above. Add the second supernatant to the first.
Part 3B: Desalting
We found from pilot studies that imidazole affects the binding curves of inverse pericams. Thus, you will continue purifying your proteins by removing any low molecular weight compounds.
- Snap off the bottom of the column, place in a 15 mL conical tube, and loosen the column's cap.
- In the large centrifuge across from the freezer, spin your columns at 1000 rcf (which is 2100 rpm for the rotor inside this centrifuge) for 2 min.
- The timing function on this centrifuge does not work! Bring your timer and manually turn the centrifuge off after 2 min. Start the timer once the rpm value approaches 2000.
- Because we all have to share one centrifuge, ideally spin with at least 2 other groups.
- When you remove your columns, the resin inside should be slanted. Make a mark right on the column where the highest point of the resin occurs, and orient that mark facing outward in the next step.
- Transfer the column to a fresh 15 mL conical tube, and then gently apply your ~ 1 mL of protein to the center of the compacted resin.
- Repeat the 2 min spin step just as before.
- Immediately after eluting your protein, transfer 10 μL of it to a clean eppendorf tube (for assaying protein concentrations), and add a 1:100 dilution of 10% BSA to the remaining protein (10 μL of BSA for ~ 1 mL of protein).
Part 4: Protein Concentration
- Prepare 12 mL Bradford reagent from the 5x concentrated stock by adding to a 9.6 mL water aliquot.
- Obtain BSA standards from the teaching faculty.
- Each tube already contains exactly 10 μL of standard (or plain water, for your blank solution).
- Add 1 mL of Bradford reagent to each standard, as well as to your three unknown protein samples. Incubate 10-20 min at room temperature.
- Measure the absorbance of each sample at 595 nm. Work as quickly as you can, because the absorbance will continue to slowly change over time. To get a sense of the error incurred due to the ongoing reaction, measure your blank sample both at the beginning and at the end of your run.
| BSA 0.1
|| Blank - start
| BSA 0.2
|| WT IPC
| BSA 0.4
|| Mutant 1
|| Mutant 2
| BSA 0.8
|| Blank - end
| BSA 1.0
For next time
- Calculate the approximate protein concentrations for your inverse pericams. First make a standard curve from the BSA data, then perform a linear fit (for example, using the Add Trendline function in Excel). The chart does not have to be especially pretty as it will not go in your report, but please do show your work, starting from the raw data. While for now mg/mL is an acceptable concentration unit, note that for your report you will want to convert to mM, μM, or nM - whichever seems most appropriate.
- The following analysis may not be included in your completed report, depending on whether your sequencing data was robust or not. We'll nevertheless take this opportunity to practice writing figures and results.
- Prepare a figure depicting the results of your diagnostic digest. Keep in mind the best practices for figures that we have discussed, from content to presentation. For example, the content should include the expected band sizes, and the presentation should include labeling of lanes as well as a few reference bands.
- Write a few sentences of results section text to accompany your diagnostic digest figure.
- Cell Lysis
- B-PER (Bacterial Protein Extraction Reagent) from Pierce
- Bovine Serum Albumin
- Protease Inibitor Set, EDTA-Free from Calbiochem
- Lysis Enzyme from Epicentre Biotechnologies
- Laemmli Sample buffer from Bio-Rad
- 2% SDS, 25% glycerol, 0.01% Bromophenol Blue in 62.5 mM Tris-HCl pH 6.8, + 5% β-mercaptoethanol just before use
- Protein Purification from Novagen/Calbiochem
- His-Bind Purification Kit buffers
- His-Bind Resin, Ni-Charged
- Zeba Desalt Spin Columns from Thermo Scientific
- Protein Concentration
- Bio-Rad Protein Assay (Bradford Reagent)