20.109(S19):Purify protein for secondary assays(Day2)

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20.109(S19): Laboratory Fundamentals of Biological Engineering

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       1. Assessing ligand binding        2. Measuring gene expression        3. Engineering biomaterials              


Introduction

To induce production of FKBP12 from the expression vector you 'cloned' in the previous laboratory session, we used the lactose-analogue isopropyl β-D-1-thiogalactopyranoside (IPTG) and arabinose to induce expression in BL21-A1 bacteria cells. The use of IPTG to induce protein expression is based on the native lac operon used for lactose metabolism in bacterial cells.

Sp17 20.109 M1D2 lac operon.png
The lac operon is composed of four genes: lacI, lacZ, lacY, and lacA. When lactose is absent, LacI (the protein encoded by lacI) binds to the operator sequence (O) upstream of lacZYA. In the presence of lactose, LacI and lactose form a complex which relieves repression of lacZYA transcription. LacZ is a β-galactosidase that cleaves lactose resulting in glucose and galactose. LacY, a β-galactoside permease, facilitates the transport of lactose across the cell membrane, and LacA, a β-galactoside transacetylase, transfers an acetyl group from acetyl-CoA to β-galactosides.

The native lac operon is a powerful tool in engineering protein expression systems because it enables researchers to control gene expression using inducer molecules. The lacZYA genes are only expressed when lactose is present. If a gene of interest is cloned downstream of the operator sequence, the expression of this gene can be controlled by LacI repression and lactose derepression. To further control the system for protein expression, IPTG is used as a lactose-analog as it is not metabolized by the cells.

Sp17 20.109 M1D2 lactose vs IPTG.png

Today you will isolate your FKBP12 protein from the bacterial cells. Remember that the FKPB12 sequence contains six histidine codons at the 5' end of the DNA sequence. 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.


You will use a nickel-agarose resin to separate your protein of interest (FKPB12) 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-A1 cells are not only producing your 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 FKBP12 by competition.

Sp17 20.109 M1D2histidine vs imidazole.png

Protocols

Part 1: Agarose gel electrophoresis of confirmation digests

Electrophoresis is a technique that separates large molecules by size using an applied electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied with this technique; agarose and acrylamide gels are the two most common sieves. The molecules to be separated enter the matrix through a well at one end and are pulled through the matrix when a current is applied across it. The larger molecules get entwined in the matrix and are stalled; the smaller molecules wind through the matrix more easily and travel farther away from the well. The distance a DNA fragment travels is inversely proportional to the log of its length. Over time fragments of similar length accumulate into “bands” in the gel. Higher concentrations of agarose can be used to resolve smaller DNA fragments.

Diagram of gel electrophoresis chamber. Larger sized DNA molecules will remain close to the well where the sample was loaded and smaller DNA molecules will migrate through the gel toward the positive electrode.

DNA and RNA are negatively charged molecules due to their phosphate backbone, and they naturally travel toward the positive electrode at the far end of the gel. Today you will separate DNA fragments using an agarose matrix. Agarose is a polymer that comes from seaweed and if you’ve ever made Jell-O™, then you already have all the skills needed for pouring an agarose gel! To prepare these gels, agarose and 1X TAE buffer (Tris base, acetic acid, and EDTA) are microwaved until the agarose is melted and fully dissolved. The molten agar is then poured into a horizontal casting tray, and a comb is added. Once the agar has solidified, the comb is removed, leaving wells into which the DNA samples can be loaded.

You will use a 1% agarose gel with SYBR Safe DNA stain (prepared by the teaching faculty) to separate the DNA fragments in your four digested samples as well as a reference lane of molecular weight markers (also called a DNA ladder).

  1. Add 5 μL of 6x loading dye to the digests.
    Illustration of proper gel loading technique.
    • Loading dye contains bromophenol blue as a tracking dye to follow the progress of the electrophoresis (so you don’t run the smallest fragments off the end of your gel!) as well as glycerol to help the samples sink into the wells.
  2. Flick the eppendorf tubes to mix the contents, then quick spin them in the microfuge to bring the contents of the tubes to the bottom.
  3. Load 25 μL of each digest into the gel, as well as 10 μL of 1kb DNA ladder.
    • Be sure to record the order in which you load your samples!
    • To load your samples, draw the volume listed above into the tip of your P200 or P20. Lower the tip below the surface of the buffer and directly over the well. Avoid lowering the tip too far into the well itself so as to not puncture the well. Expel your sample slowly into the well. Do not release the pipet plunger until after you have removed the tip from the gel box (or you'll draw your sample back into the tip!).
  4. Once all the samples have been loaded, attach the gel box to the power supply and electrophorese the gel at 125 V for 45 minutes.

Part 2: Lyse BL21-A1 pET21_FKBP12 cells

Previously, the teaching faculty inoculated 5 mL of LB media with BL21-A1 pET21_FKBP12, incubated the culture at 37 °C for 7 hours and then stored at 4 °C overnight. The overnight culture was used to inoculate 50 mL of fresh LB media containing ampicillin at a 1:10 dilution. When the OD600 of this culture reached 0.5-0.8, IPTG was added to a final concentration of 1 mM and arabinose to a final concentration of 0.2% were added to the E. coli bacterial culture to induce production of FKBP12.

The induced culture was incubated for ~16 hr at 25°C then the cells were collected by centrifugation at 3000 g for 10 min. The harvested cells were stored in the -80 °C freezer.

  1. Retrieve your BL21-A1 pET21_FKBP12 cell pellet from the front laboratory bench and leave it on your bench to thaw.
    • You will also obtain a cell pellet of uninduced cells that was prepared by the teaching faculty as a control to examine the IPTG / arabinose induction step.
  2. Prepare 1.5 mL of lysis buffer.
    • Use the information in the table below to calculate the volume of each stock reagent that is needed to prepare the lysis buffer with the specified final concentrations.
  3. Confirm your calculations with the teaching faculty, then prepare your lysis buffer solution.
Reagent (stock concentration) Final concentration of stock reagent in lysis buffer Volume of stock reagent in lysis buffer
Tris, pH = 7 (1 M) 50 mM
NaCl (1 M) 150 mM
Glycerol (40%) 10%
DTT (1 M) 1 mM
AEBSF (100 mM) 1 mM
H2O add for a total of 1.5 mL of lysis buffer
  1. Weigh each cell pellet using the balance and add 1 mL of lysis buffer per 1 g of cell pellet.
    • Note: first, you'll need to weigh a 50 mL conical tube.
  2. Resuspend each pellet completely in the lysis buffer then transfer the cell suspension to a 2 mL eppendorf tube.
    • Note: You may need to cut the tip off of a P1000 pipette tip to transfer cell suspension.
  3. Add lysozyme (stock concentration of 50 mg/mL) to each cell suspension such that the final concentration is 300 μg/mL.
  4. Incubate in the 4 °C cooler for 1 h on the nutator.

Part 3: Prepare Ni-NTA affinity column

  1. Set up one purification column using the stand and clamp at your bench according to the example at the front bench.
  2. Obtain a 500 μL aliquot of 50% slurry (Ni-NTA resin) from the front laboratory bench, mix the slurry by inverting the tube several times and pipette the slurry into the column.
  3. Wash the slurry with 3 mL of 1X PBS by pipetting buffer over the top of the settled beads in the column.
  4. Allow the PBS drip out of the column by gravitational flow, putting a stopper at the spout (bottom) of the column just before the PBS has completely flowed through the column.
  5. Leave the column on your benchtop until you are ready to add the cell lysate in Part 3.

Part 4: Purify FKBP12 protein

  1. Retrieve your lysed cell pellets from the 4 °C cooler.
  2. Transfer 15 μL from each tube (-IPTG and +IPTG) into labeled 1.5 mL eppendorf tubes and give the aliquots to the teaching faculty.
    • You will use these aliquots to examine protein yield prior to purification using polyacrylamide gel electrophoresis (PAGE) on a later date.
  3. Calculate the volume of 1 M MgCl2 to add for a final concentration of 10 mM in your remaining cell lysate.
  4. Add the volume of MgCl2 you calculated in the previous step and 10 μL of DNase to the tube that contains the IPTG-induced sample.
    • You will only complete the purification protocol for the IPTG-induced sample!
  5. Incubate for 30 min in the 4 °C cooler on the nutator.
  6. Centrifuge your sample for 30 min at 16,000 rcf in the 4 °C cold room.
    • Alert the teaching faculty when you are ready to centrifuge your sample and you will be escorted to the cold room.
  7. Check that the supernatant in your sample is clear with little to no 'cloudiness'.
  8. Load the supernatant from your protein solution onto the column.
    • Make sure the stopper is on the column when loading the supernatant.
  9. Add a cap to the top of your purification column.
    • At this point the column should be sealed on both sides.
  10. Incubate the column with the Ni-NTA resin and bacterial lysate at 4 °C on the nutator for 30 min to promote binding.
  11. Retrieve your column and reposition it on the clamp at your bench.
  12. Obtain an aliquot of PBS buffer containing 10 mM imidazole from the front laboratory bench.
  13. Wash the column by adding 1.5 mL of the PBS buffer containing 10 mM imidazole to the column and save the first ~500 μL of each wash in a well labeled microcentrifuge tube. The remainder of the wash can be discarded.
  14. Repeat Step #13 a total of three times.
  15. Elute your FKBP12 protein by adding 1 mL of PBS buffer containing 250 mM imidazole to the column.
    • Carefully collect all of the elution flow through in a well labeled microcentrifuge tube.
  16. Repeat Step #15 a total of two times.
  17. Give your three wash fractions, your two protein elutions, and your used purification column to the teaching faculty.
    • Be sure that all tubes are clearly labeled with your team color and section!

Reagents

For gel electrophoresis:

  • 1% agarose in water, with 10 uL SYBR Safe DNA gel stain per 100 uL agarose solution
  • 1X TAE gel electrophoresis buffer: (BioRad)
    • 40 mM Tris
    • 20 mM acetic acid
    • 1 mM EDTA
  • 6X gel loading dye, blue (NEB)
  • 1 kb DNA ladder (NEB)

For protein induction / purification:

  • LB (Luria-Bertani broth, BD Biosciences)
    • 1% Tryptone
    • 0.5% Yeast Extract
    • 1% NaCl
    • autoclaved for sterility
  • Ampicillin stock (sigma): 100 mg/mL, aqueous, sterile-filtered, store at -20 °C
  • PBS: phosphate saline buffer (VWR)
  • Ni-NTA agarose (Qiagen)
  • AEBSF: 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma)
  • Lysozyme from chicken egg white (Sigma)
  • DNase I (1 unit/uL, Sigma)
  • DTT : DL-dithiothreitol (Sigma)
  • imidazole (Sigma)
  • Tris (Sigma)
  • NaCl (Sigma)

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