BE.109:Protein engineering/Purifying beta-galactosidase

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BE.109 Laboratory Fundamentals of Biological Engineering

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Contents

Introduction

It’s time to confess. You’ve been misled about the protein that you’re working with during this experimental module. More precisely, you’ve been incompletely informed. The truth is this: the beta-galactosidase gene has been modified to add six histidine residues at the amino-terminus of the protein. That’s it. Now you know. The reason they are there is to help with today’s purification of the enzyme since (1) very few other proteins in the cell will have six histidine residues in a row (2) histidine has an affinity for metals such as zinc and nickel, a useful property that can be exploited as a purification scheme, and (3) proteins are modular so it’s a good guess that the beta-galactosidase enzyme will still fold and function despite the “6xHis tag” at its N-terminus. We should have told you sooner.

Protein purification is fundamentally an enrichment process, an experiment designed to keep the protein of interest and eliminate other proteins from the sample. Proteins can be separated based on their physical characteristics. For example, “gel filtration” is a method that separates proteins by size, with small proteins collected in one tube and larger proteins in another. Alternatively, proteins can be separated by their charge or their affinity for particular molecules. The latter is the method you will use today but be aware that many purification schemes require multiple “rounds” of purification, separating proteins first by size then by charge, for example, to more completely eliminate proteins that are not desired in your final sample. With each step, however, some of the desired protein is invariably removed or destroyed so schemes must balance yield with purity, aiming to enrich to as great a degree as possible, losing as little material as possible, and maintaining the protein in its active form as best as possible. No small task.

We are all familiar with metal-protein complexes (for example hemoglobin is an iron-protein complex) but it was a clever and insightful moment when researchers thought to use this property of proteins as a tool for purification. Metals can be immobilized on a solid support, like an agarose bead or a ceramic resin, which is then mixed with a cell lysate containing the metal-binding protein. The metal-binding protein will “stick” to the solid support and the unbound proteins can be removed in the supernatant. The protein of interest can be “eluted” from the resin using a competitor that has even higher affinity for the metal. With the tools of DNA engineering, any gene can be modified to express affinity tags, short sequences with high affinity for a known ligand, making this metal-binding strategy more generally useful. In your case, you’ll be using the 6xHis-tag to bind beta-galactosidase to a nickel-agarose resin, and then you’ll be competing the protein off the resin with imidazole, a compound similar to histidine’s ringed chemical structure.

Image:Macintosh HD-Users-nkuldell-Desktop-Ni-agarose purif.png

Images from Qiagen protocol for Ni-NTA protein purification kit.

In today’s experiment, you will isolate beta-galactosidase from the cells you froze last time. After lysing the cells and purifying the enzyme from the cell lysate with Ni-agarose, you will measure the concentration of purified protein using a BioRad Assay and measure the enzyme’s activity using a beta-galactosidase assay. If we had more time it would be interesting to run the purified proteins on a protein gel to observe how many proteins remain in the sample after purification. This would also help us understand the consequences of introducing the unnatural amino acid into the enzyme.

Protocol

Part 1: Cell Lysis

Although soap, bleach or other harsh chemicals could be used to lyse the cells, these treatments would also denature the cell’s proteins so instead you will use a gentle, enzymatic treatment to open the bacterial bag. The solution is a commercial reagent available through a company called Epicentre.

  1. Retrieve your “plus IPTG” sample and the “plus IPTG plus aa” sample from the freezer. Resuspend each of the bacterial pellets in 200 μl of the Lysis Solution provided by the teaching faculty.
  2. Incubate the tubes at room temperature for 5 minutes.
  3. Pellet the cell debris by spinning the tubes in a microfuge for 2 minutes at 13,000 RPM.
  4. Transfer the supernatant to two clean, labeled eppendorf tubes and move them to an ice bucket. Your protein solutions should be kept on ice as much as possible.

Part 2: Purification

There are three steps to this purification protocol: binding the lysate to the resin, washing the resin to remove the unbound or loosely bound proteins, and finally eluting the protein off the resin. The solutions needed for each step (sensibly called Binding Buffer, Wash Buffer and Elution Buffer) have progressively higher concentrations of imidazole. Before you begin the purification, you need to equilibrate the resin in Binding Buffer to prepare it for incubation with the cell lysate.

Two protocols are offered below. The first is a more standard protocol with which you can purify your his-tagged beta-galactosidase through its affinity for Ni-agarose. The second is a newer protocol that has yet to be optimized. It uses paramagnetic beads that are coated with an antibody that recognizes the his-tag. This second protocol may be fun to try. Also, the startup company that has offered us the reagent has expressed interest in the protocol comparison done by our class. Pick whichever protocol you would prefer to try, and carefully document your work (as always). Later you will post your results to the discussion page so Sunrise Science Products [1] can examine them.

Protocol for Ni-agarose purification

  1. Prepare the resin: Flick the stock bottle to resuspend the slurry. Use your P1000 to remove 0.5 ml of resin. Spin in a microfuge at 5000 rpm (not full speed!) for one minute. Use your P1000 to remove the supernatant to a waste-collecting tube, eg a 15 ml conical tube, and add 500 μl Binding Buffer. Invert the tube to resuspend the resin in the buffer then spin the tubes again at 5000 rpm for one minute. Remove the supernatant once more and add 500 ul Binding Buffer.
  2. Bind the lysate to the resin: Mix 100 μl of your cell lysate with 700 μl Binding Buffer and 200 μl of the prepared resin, inverting the tube with the resin to resuspend it before removing your aliquot. Place the eppendorfs on a nutator at room temperature for 10 minutes. Spin the tubes in a microfuge at 5000 rpm for one minute. Remove and discard the supernatant. A more conservative (and less time-pressed) researcher would save this supernatant but you should consider what proteins there could be in the discarded sample. You will be asked about them as part of your “FNT” assignment.
  3. Wash the resin: Add 0.5 ml of Wash Buffer. Invert the tubes to resuspend the resin. Spin the tubes in a microfuge at 5000 rpm for one minute. Remove and discard the supernatant.
  4. Elute the protein from the resin: Add 200 μl of Elution Buffer to the resin and flick to mix. Place the eppendorfs on a nutator at room temperature for five minutes. Make sure the small volume of liquid in the tubes is moving while the tubes are on the nutator and flick each mix after 2.5 minutes to fully resuspend them again. Spin the tubes at 5000 rpm for one minute. Move the supernatant into two clean eppendorf tubes, leaving the resin behind. There should be more than 100 ul but less than 200 ul of each sample. These are your purified fractions that you will examine for protein content and enzymatic activity.

Protocol for anti-his paramagnetic bead purification

  1. Prepare the beads: Flick the stock bottle to resuspend the slurry. Use your P1000 to remove 0.1 ml of antibody coated paramagnetic beads to two eppendorf tubes. Place the tubes in the magnetic strip provided and you should see the black beads drawn to the side of each tube. Leave the tubes in the strip to pipet the liquid out of the eppendorf tubes and into to a waste-collecting tube, eg a 15 ml conical. Add 200 μl Binding Buffer. Remove the tubes from the magnetic strip and flick them to resuspend the beads in the buffer. Place the tubes back in the strip, remove the supernatant once more and add 900 ul Binding Buffer to each.
  2. Bind the lysate to the beads: Mix 100 μl of your cell lysate with the 900 μl of prepared beads, inverting the tube to resuspend. Place the eppendorfs on a nutator at room temperature for 10 minutes. Place the tubes in the paramagnetic strip to remove and discard the supernatant. A more conservative (and less time-pressed) researcher would save this supernatant but you should consider what proteins there could be in this discarded sample. You will be asked about them as part of your “FNT” assignment.
  3. Wash the beads: Add 0.5 ml of Wash Buffer. Invert the tubes to resuspend the resin. Place the tubes in the magnetic strip then remove and discard the supernatant from each.
  4. Elute the protein from the beads: Add 200 μl of Elution Buffer to the beads and flick to mix. Place the eppendorfs on a nutator at room temperature for five minutes. Make sure the small volume of liquid in the tubes is moving while the tubes are on the nutator and flick each mix after 2.5 minutes to fully resuspend them again. Place the tubes in the magnetic strip then move the supernatant into two clean eppendorf tubes, leaving the resin behind. There should be more than 100 ul but less than 200 ul of each sample. These are your purified fractions that you will examine for protein content and enzymatic activity.

Part 3: BioRad Assay of Purified Proteins

Refer back to the protocol you performed on Day 1 of this module. The following table is offered to help you organize your work. You should use 100 ul of your purified fractions for this assay.

Tube Contents A595 Tube Contents A595
0 Blank 0 undiluted beta-galactosidase
0.1 BSA 1:5 beta-galactosidase
0.2 BSA 1:10 beta-galactosidase
0.4 BSA
0.6 BSA + 100 ul Purified fraction +IPTG
0.8 BSA ++ 100 ul Purified fraction +IPTG+aa
1 BSA

Part 4: Beta-galatosidase Assay of Purified Proteins

Refer back to the protocol you performed on Day 1 of this module. The following table is offered to help you organize your work but you should prepare one that is to your liking if this isn’t.

Tube Enzyme Start Stop A420
0 none 0:00 0
1 1:10 b-gal stock 0:10
2 1:10 b-gal stock 0:20
3 1:10 b-gal stock 0:30
4 1:100 b-gal stock 0:40
5 1:100 b-gal stock 0:50
6 1:100 b-gal stock 1:00
7 10 μl of +IPTG purif 1:10
8 10 μl of +IPTG purif 1:20
9 10 μl of +IPTG purif 1:30
10 10 μl of +IPTG+aa purif 1:40
11 10 μl of +IPTG+aa purif 1:50
12 10 μl of +IPTG+aa purif 2:00

For Next Time

  1. Imagine you’re an investigator studying protein chemistry at MIT and a UROP comes to see you with a great experimental idea they’d like to try over IAP: to purify beta-galactosidase based on its affinity for lactose. “Since the enzyme binds the sugar, all we’d have to do is bind the sugar to some resin and then run cell extracts with the protein over the beads,” they suggest. What would you say to this student? Please include a few scientific points.
  2. Sadly we don’t have time to run an SDS-PAGE of the protein fractions from your purification. Instead, you should sketch what the gel would look like, using an arrow at the side of your drawing to indicate which band is beta-galactosidase. The lanes to include are listed below. To get you started, the first three lanes are the same as the samples you ran on your protein gel last time.
    • Lane 1 = molecular weight markers
    • Lane 2 = “no IPTG” cells
    • Lane 3 = “plus IPTG” cells
    • Lane 4 = lysate of “plus IPTG” cells before incubation with the resin
    • Lane 5 = discarded supernatant from “plus IPTG” lysate after incubation with the resin, assuming 100% efficient binding of His6-tagged protein to the resin.
    • Lane 6 = purified fraction after elution of “plus IPTG” lysate from resin
  3. Prepare a standard curve using the BSA standards, showing the best-fit line and r-squared value, and then use the equation to determine the concentration of protein in each of your purified fractions. Please include one hand written calculation we can check your work. Describe how confident you are in the values you measured for your purified fractions.
  4. Use the data from today’s beta-galactosidase assays to calculate the activity of your purified fractions of protein. Please include one hand-written sample calculation so we can check your work. Compare the activity of the stock, and the purified proteins, saying what you can about
    1. the induction of the enzyme with and without the unnatural amino acid
    2. the purification of the enzyme with and without the unnatural amino acid
    3. the activity of the enzyme with and without the unnatural amino acid
    • This is your chance to pull your understanding of this work together. Show off a little here, take a chance, propose something you’d like to try next.
  5. Post your data to the discussion page of this protocol so others can consider it. A table has been started to organize the class data.
  6. Please download a midsemester evaluation form Image:Macintosh HD-Users-nkuldell-Desktop-BE109-BE109 S06class-BE109 MidsemEvalS06.doc. Complete the questionnaire and then print it out without including your name to turn in next time. If there is something you'd like to see done differently for the rest of the course, this is your chance to lobby for that change. Similarly, if there is something you think the class has to keep doing, let us know that too.
  7. Prepare a ten-minute oral presentation of a primary research paper related in topic to the experiments performed in this experimental. Some articles that are suitable for presentation are listed under the link for Module 2 Day 4. These can be reserved on a first come/first served basis so email your choice as soon as you’ve decided. Alternatively, you’re also welcome to present a research idea stemming from the experiments you have performed in Module 2.

Reagents List

Lysis Buffer “EasyLyse Bacterial Protein Extraction Solution” from Epicentre

  • 10 mM Tris, pH7.5
  • 1 mM EDTA
  • 1% non-denaturing detergent
  • 2 mM MgCl2
  • secret “enzymatic mix” (likely lysozyme)

Resin Binding Buffer

  • 50 mM NaH2PO4, pH8
  • 0.5 M NaCl
  • 10 mM imidazole

Resin Wash Buffer

  • 50 mM NaH2PO4, pH8
  • 0.5 M NaCl
  • 20 mM imidazole

Resin Elution Buffer

  • 50 mM NaH2PO4, pH8
  • 0.5 M NaCl
  • 250 mM imidazole
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