Difference between revisions of "20.109(S21):M3D3"

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(Part 3: Purify IPC variants)
(Part 3: Purify IPC variants)
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explain expression system components...
 
explain expression system components...
  
===Part 3: Purify IPC variants===
+
===Part 3: Purify IPC protein===
  
 
'''Induce expression of IPC'''
 
'''Induce expression of IPC'''
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#Incubate at 25 °C with shaking at 100 rpm overnight.
 
#Incubate at 25 °C with shaking at 100 rpm overnight.
 
#To harvest the cells, centrifuge the culture at 3000 g for 15 min at 4 °C.
 
#To harvest the cells, centrifuge the culture at 3000 g for 15 min at 4 °C.
#Cell pellets were stored at -80 °C until used for purification.
+
#Cell pellet was stored at -80 °C until used for purification.
  
 
<font color =  #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:
 
<font color =  #4a9152 >'''In your laboratory notebook,'''</font color> complete the following:
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'''Lyse BL21(DE3)pLysS cells expressing pRSET_IPC''
 
'''Lyse BL21(DE3)pLysS cells expressing pRSET_IPC''
  
#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.  
+
#Obtain a 2 mL aliquot of room temperature BugBuster buffer and the induced BL21(DE3)pLysS pRSET_IPC cell pellet.
#*When you are ready to begin, add 1:1000 of cold nuclease enzyme (obtained from teaching staff) to the BugBuster solution.  
+
#*BugBuster is a bacterial lysis and protein extraction solution, which contains 0.1% bovine serum albumin and 1:200 protease inhibitor cocktail to guard against protein degradation.  
#Per cell pellet (4 total), add the appropriate volume of enzyme-containing BugBuster and resuspend by pipetting until the solution is relatively homogeneous.
+
#*Add 1:1000 of cold nuclease enzyme to the BugBuster buffer.  
#*Resuspend -IPTG samples in 300 &mu;L, and +IPTG samples in 600 &mu;L - do you remember why?
+
#Add 600 &mu;L of the BugBuster with nuclease enzyme to the BL21(DE3)pLysS pRSET_IPC cell pellet.
#Pipet up and down to mix.
+
#Resuspend the cell pellet by pipetting until the solution is homogeneous.
#*Be sure the sample is homogenous.  You will likely need to pipet up and down 15-20x.
+
#Incubate on the nutator at room temperature for 10 minutes.
#Incubate the solutions (at room temperature) for 10 minutes on the nutator.
+
#Centrifuge the lysed cell suspension for 10 minutes at maximum speed.  
#*During this incubation, you may begin the resin preparation described in Part 3.
+
#Transfer the supernatant to a fresh microcentrifuge tube.
#Finally, centrifuge for 10 minutes 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.
+
'''Prepare Ni-NTA affinity column'''
 +
 
 +
 
 +
'''Purify TDP43-RRM12 from cell lysate'''
 +
 
 +
 
 +
'''Remove imidazole from purified IPC'''
  
 
===Part 2: Advance preparation for SDS-PAGE of protein extracts===
 
===Part 2: Advance preparation for SDS-PAGE of protein extracts===

Revision as of 17:53, 5 February 2021

20.109(S21): Laboratory Fundamentals of Biological Engineering

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Spring 2021 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Antibody engineering        M2: Drug discovery        M3: Protein engineering       


Introduction

Now that you have prepared DNA encoding your mutant inverse pericams, we need to produce the proteins so we can assess the affinity and cooperativity of your new protein. Since the last time you were here, your oh-so devoted teaching staff transformed competent bacteria (strain NEB 5α) with your SDM product. Successfully transformed NEB 5α bacteria grew into colonies on ampicillin-containing plates, and two colonies were picked to grow in liquid cultures. The liquid culture of E. coli serves as a plasmid-generating factory. Today you will purify the plasmids carrying the hopefully mutated inverse pericam gene using a Qiagen mini-prep kit. To ensure that the SDM reaction was successful, and therefore the IPC gene was mutated, you will prepare the purified DNA for sequencing analysis. Though the NEB 5α cell strain is able to replicate the inverse pericam plasmid DNA, it cannot produce the inverse pericam protein. Therefore, you will transform your IPC mutant plasmids into a new bacterial system, BL21(DE3)pLysS, that can produce the protein for later analysis.

Vector map of pRSET modified from Invitrogen manual.

The bacterial expression vector we are using, pRSET, encodes many features that make it ideal for our research purposes. To enable selection of bacterial cells that carry the plasmid, an antibiotic cassette (specifically, an ampicillin marker) is included on the vector. Several features are included to promote protein expression and purification. In the image to the right a schematic representation of these features is shown. The T7 promoter drives expression of the gene that encodes IPC (or your mutated IPC). To ensure that the transcript is translated into a protein, a Ribosome Binding Site (RBS) is included. The ATG sequence serves as the transcriptional start and the 6xHis represents the six-histidine residue tag that is used for protein purification via affinity chromatography.

As mentioned above, pRSET encodes the bacteriophage T7 promoter, which is active only in the presence of T7 RNA polymerase (T7RNAP), an enzyme that therefore must be expressed by the bacterial strain used to make the protein of interest. We will use the BL21(DE3)pLysS strain, which has the following genotype: F-, ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR). In BL21(DE3), T7RNAP is associated with a lac construct. Constitutively expressed lac repressor (lacI gene) blocks expression from the lac promoter; thus, the polymerase will not be produced except in the presence of repressor-binding lactose or a small-molecule lactose analogue such as IPTG (isopropyl β-D-thiogalactoside). To reduce ‘leaky’ expression of the protein of interest (in our case, inverse pericam), the pLysS version of BL21(DE3) contains T7 lysozyme, which inhibits basal transcription of T7RNAP. This gene is retained by chloramphenicol selection, while the pRSET plasmid itself (and thus inverse pericam) is retained by ampicillin selection.

Overview of protein expression system.


After completing mini-preps to isolate your plasmid DNA today (two mutant pRSET-IPC candidates), you will prepare the DNA for sequencing analysis, as well as use it immediately for transformation. In order to transform BL21(DE3)pLysS cells with your mutant IPC plasmids, you will first have to make the cells competent, i.e., able to efficiently take up foreign DNA. With the NEB 5α strain, we used commercially available competent cells that did not need further treatment prior to DNA addition. Today, you will make chemically competent cells yourself using calcium chloride, then incubate them with plasmid DNA and heat shock them as before prior to plating. Whether prepared by a company or by you, remember that competent cells are extremely fragile and should be handled gently, i.e. kept cold and not vortexed. Bacterial transformation is efficient enough for most lab purposes, resulting in as many as 109 transformed cells per microgram of DNA, but even with highly competent cells only 1 DNA molecule in about 10,000 is successfully transformed.

After today's lab session, the teaching staff will pick colonies and set up liquid overnight cultures from your transformed BL21(DE3)pLysS cells. Next time, you will add IPTG to these liquid cultures to induce expression of your mutant proteins, which you will then isolate and characterize.

Protocols

Part 1: Participate in Comm Lab workshop

Our communication instructors, Dr. Prerna Bhargava and Dr. Sean Clarke, will join us today for a discussion on preparing a Research proposal presentation.

Part 2: Prepare protein expression system

using BL21(DE3)pLysS chemically competent cells...

pRSET_IPC and pRSET_IPC variants transformed using heat shock as on M1D3...

explain expression system components...

Part 3: Purify IPC protein

Induce expression of IPC

  1. Inoculate 5 mL of LB media containing 50 μg/mL ampicillin with a colony of BL21(DE3)pLysS cells transformed with pRSET_IPC.
  2. Incubate the culture overnight at 37 °C with shaking at 220 rpm.
  3. Dilute the overnight culture 1:10 in 50 mL of fresh LB media containing 50 μg/mL ampicillin and 34 μg/mL chloramphenicol.
  4. Incubate at 37 °C until the OD600 = ~0.6 with shaking at 220 rpm, approximately 4 hours.
  5. To induce IPC protein expression, add IPTG to a final concentration of 1 mM.
  6. Incubate at 25 °C with shaking at 100 rpm overnight.
  7. To harvest the cells, centrifuge the culture at 3000 g for 15 min at 4 °C.
  8. Cell pellet was stored at -80 °C until used for purification.

In your laboratory notebook, complete the following:

  • Why is it important that both ampicillin and chloramphenicol are added to the growth media?

'Lyse BL21(DE3)pLysS cells expressing pRSET_IPC

  1. Obtain a 2 mL aliquot of room temperature BugBuster buffer and the induced BL21(DE3)pLysS pRSET_IPC cell pellet.
    • BugBuster is a bacterial lysis and protein extraction solution, which contains 0.1% bovine serum albumin and 1:200 protease inhibitor cocktail to guard against protein degradation.
    • Add 1:1000 of cold nuclease enzyme to the BugBuster buffer.
  2. Add 600 μL of the BugBuster with nuclease enzyme to the BL21(DE3)pLysS pRSET_IPC cell pellet.
  3. Resuspend the cell pellet by pipetting until the solution is homogeneous.
  4. Incubate on the nutator at room temperature for 10 minutes.
  5. Centrifuge the lysed cell suspension for 10 minutes at maximum speed.
  6. Transfer the supernatant to a fresh microcentrifuge tube.

Prepare Ni-NTA affinity column


Purify TDP43-RRM12 from cell lysate


Remove imidazole from purified IPC

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).

Reagents list

  • QIAprep Spin Miniprep Kit reagents
  • Sequencing primers (concentration = 5 pmol/μL)
  • 100 mM CaCl2, sterile
  • LB (Luria-Bertani broth)
    • 1% Tryptone
    • 0.5% Yeast Extract
    • 1% NaCl
    • autoclaved for sterility
  • Ampicillin stock: 100 mg/mL, aqueous, sterile-filtered, store at +4 °C
  • Chloramphenicol stock: 34 mg/mL in ethanol, store at -20 °C
  • LB+AMP+CAM plates
    • LB with 1.5% agar and 100 μg/mL ampicillin and 34 μg/mL chloramphenicol


  • IPTG (isopropyl β-D-1-thiogalactoside), 0.1 M


  • 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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