20.109(S16):Purify protein (Day6)

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20.109(S16): Laboratory Fundamentals of Biological Engineering

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Introduction

Last time you used the lactose-analogue IPTG to induce expression of inverse pericam in BL21(DE3)pLysS bacteria. Today you will isolate your mutated IPC and the wild-type IPC from the bacterial cells. You will also begin characterizing your wild-type and mutant proteins.

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 (IPC) 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 1 can be washed away. Remember, the BL21(DE3)pLysS cells are not only producing our protein, but also the proteins needed for cellular function and survival. 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.

Histidine
Imidazole

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 dodecyl 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 a protein band at the expected molecular weight for inverse pericam (47 kDa + 3 kDa from the His-tag = 50 kDa), which may be very faint or non-existent in the -IPTG control sample, but bright and thick in the +IPTG 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.

To measure the concentration of your purified protein, you will use the Pierce Micro BCA Protein Assay Reagent Kit. This kit enables colorimetric detection and quantification of the total protein within a sample. The ability to measure protein concentration is based on the detection of Cu1+ by the detection reagent, bicinchoninic acid (BCA). The Cu1+ is formed when Cu2+ is reduced by protein in an alkaline environment. Through this reduction reaction, a purple product is formed by the chelation of BCA and Cu1+ at a 2:1 ratio. This water-soluble complex has a strong absorbance at 562 nm. Because absorbance and protein concentration have a linear relationship, it is possible to compare the absorbance of an unknown protein sample to a standard curve, generated with samples of known protein concentrations, and calculate the concentration of protein in the experimental sample.

Protocols

Part 1: Lysis of cells producing wild-type and mutant IPC

  1. You will be given a 2 mL aliquot of room temperature BugBuster buffer (bacterial lysis and protein extraction solution), which contains 0.1% bovine serum albumin (BSA, a stabilizer) and 1:200 protease inhibitor cocktail to guard against protein degradation.
    • When you are ready to begin, add 1:1000 of cold nuclease enzyme (obtained from teaching staff) to the BugBuster solution.
  2. Per cell pellet (4 total), add the appropriate volume of enzyme-containing BugBuster 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?
  3. Pipet up and down to mix.
    • Be sure the sample is homogenous. You will likely need to pipet up and down 15-20x.
  4. Incubate the solutions (at room temperature) for 10 minutes on the nutator.
    • During this incubation, you may begin the resin preparation described in Part 3.
  5. Finally, centrifuge for 10 minutes at maximum speed and transfer supernatants to fresh tubes.
  6. While one partner completes Part 2, the other partner can begin/continue with the resin preparation in Part 3.

Part 2: Advance preparation for SDS-PAGE of protein extracts

  1. Last time you measured the amount of cells in each of your samples (-IPTG and +IPTG of the wild-type IPC and one correct mutant). (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.
  2. For your other three 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.
  3. 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.
  4. Finally, add 3 μL of 6X sample buffer to 15 μL of each of your diluted lysates. These will be stored in the freezer until next time.
Sample Name OD600 Sample Volume (μL) Water Volume (μL) Total Volume (μL)
-IPTG WT 15
+IPTG WT 15
-IPTG mutant 15
+IPTG mutant 15

Part 3: Protein purification

Part 3A: Nickel-agarose purification

You will process two samples (+IPTG wild-type and +IPTG mutant IPC) according to the following procedure. Keep all buffers on ice when not in use. All spins should be performed at 1000 rcf (3300 rpm) for 1 minute.

  1. The following buffers are aliquoted and located at the front bench:
    • Ni-NTA His-bind resin
    • 1X Ni-NTA Bind Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole)
    • 1X Ni-NTA Wash Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 20 mM imidazole)
    • 1X Ni-NTA Elute Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 250 mM imidazole)
    • Note: Two special waste streams should be created for this affinity purification procedure, (1) nickel waste for the 50% slurry, and (2) imidazole waste for the Bind, Wash, and Elute Buffers.
  2. Gently mix the Ni-NTA His-bind resin to fully resuspend it, then distribute 400 μL of the resin to each of two 2 mL centrifuge tubes.
    • Label one tube as wild type and the other as mutant.
  3. Add 1.6 mL (2 x 800 μL) of 1X Ni-NTA Bind Buffer to the Ni-NTA His-bind resin.
    • Resuspend the resin by pippeting the solution up and down several times (10-15), then centrifuge (see conditions above).
  4. Carefully remove the supernatant and discard it in the appropriate waste stream.
  5. Add your cleared cell lysate from Part 1 to the resin, then put your tubes on the nutator at 4°C for 30 minutes.
    • Be sure to add the wild type and mutant lysates to the correct tubes!
  6. Centrifuge (see conditions above).
  7. Remove the supernatent and discard it in the appropriate waste stream.
  8. Add 1 mL of 1X Ni-NTA Wash Buffer to the resin.
  9. Centrifuge (see conditions above).
  10. Remove the supernatent and discard it in the appropriate waste stream.
  11. Repeat Steps #8-10.
  12. 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" or “pure IPC WT.”
  13. Do not throw away the resin yet either! Instead, repeat Step #12 one more time and add the supernatant to the sample collected in Step #12. You'll have a total of 1 mL of pure wild-type and 1 mL of pure mutant IPC.

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.

  1. For each of your two samples, snap off the bottom of a Zeba column, place in a 15 mL conical tube, and loosen the column's cap.
  2. In the large centrifuge in the cold room across the hall, spin your columns at 1000 rcf (which is 2100 rpm for the rotor inside this centrifuge) for 2 minutes.
    • Because we all have to share one centrifuge, ideally spin with at least 2 other groups.
  3. 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.
  4. Repeat the 2-minute spin step just as before.
  5. Immediately after eluting your protein, transfer 10 μL of purified protein to a clean eppendorf tube for assaying protein concentrations (Part 4). Also take 15 μL to a separate eppendorf tube for SDS-PAGE analysis and add 3μL of 6X SDS-PAGE loading dye(give this tube to your instructor).
  6. Then 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

Part 4A: Prepare diluted albumin (BSA) standards

  1. Obtain a 0.25 mL aliquot of 2.0 mg/mL albumin standard stock and a conical tube of diH2O from the front bench.
  2. Prepare your standards according to the table below using dH2O as the diluent:
    • Be sure to use 5 mL polystyrene tubes found on the instructors bench when preparing your standards as the volumes are too large for the microcentrifuge tubes.
Vial
Volume of diluent (mL) Volume (mL) and source of BSA (vial) Final BSA concentration (μg/mL)
A 2.25 0.25 of stock 200
B 3.6 0.4 of A 20
C 2.0 2.0 of B 10
D 2.0 2.0 of C 5
E 2.0 2.0 of D 2.5
F 2.4 1.6 of E 1
G 2.0 2.0 of F 0.5
H 4.0 0 Blank

Part 4B: Prepare Working Reagent (WR) and measuring protein concentration

  1. Use the following formula to calculate the volume of WR required: (# of standards + # unknowns) * 1.1 = total volume of WR (in mL).
  2. Prepare the calculated volume of WR by mixing the Micro BCA Reagent MA, Reagent MB, and Reagent MC such that 50% of the total volume is MA, 48% is MB, and 2% is MC.
    • For example, if your calculated total volume of WR is 100 mL, then mix 50 mL of MA, 48 mL of MB, and 2 mL of MC.
    • Prepare your WR in a 15 mL conical tube.
  3. Pipet 0.5 mL of each standard prepared in Part 4A into clearly labeled 1.5 mL microcentrifuge tubes.
  4. Prepare your protein samples by adding 990 μL of dH2O to your 10 μL aliquot of purified protein, for a final volume of 1 mL in clearly labeled 1.5 mL microcentrifuge tubes.
  5. Add 0.5 mL of the WR to each 0.5 mL aliquot of the standard and to your 0.5 mL protein samples.
  6. Cap your tubes and incubate at 60°C in the water bath for 1 hour. During this time download the sample data on the Discussion page to practice estimating protein concentration of your samples.
  7. Following the incubation, the teaching faculty will use the spectrophotometer to measure the protein concentrations of your standards and your purified samples.
    • The cuvette filled only with water (H) will be used as a blank in the spectrophotometer.
    • The absorbance at 562 nm for each solution will be measured and the results will be posted to today's Discussion page.
    • Establish your standard curve by plotting OD562 for each BSA standard (B-H) vs. its concentration in μg/mL.
    • Use the standard curve in its linear range (0.5 - 20 μg/mL), and its linear regression in Excel, to determine the protein concentration of each unknown sample (wild-type and mutant IPC).

Reagent list

  • BugBuster Protein Extraction Reagent from EMD Millipore
    • 0.1% BSA
    • 1:200 protease inhibitors
    • 1:1000 nuclease enzyme
  • 6X Laemmli sample buffer from Boston BioProducts
    • 2% SDS, 6% glycerol, 0.03% Bromophenol Blue in 375 mM Tris-HCl pH 6.8, + 9% β-mercaptoethanol
  • Protein purification supplies from Novagen/Calbiochem
    • Ni-NTA His-Bind Resin
    • 1X Ni-NTA Bind Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole)
    • 1X Ni-NTA Wash Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 20 mM imidazole)
    • 1X Ni-NTA Elute Buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 250 mM imidazole)
  • Zeba Desalt Spin Columns from Thermo Scientific
    • 7000 Da MW cut-off
  • Micro BCA Protein Assay Kit from Thermo Scientific
    • Micro BCA Reagent A (MA)
    • Micro BCA Reagent B (MB)
    • Micro BCA Reagent C (MC)
    • Bovine Serum Albumin Standard, 2 mg/mL

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