Justification of procedures:

To test the protease efficacy, we chose to run an SDS-page as we did not have access to a nanodrop. We ran a trial Bradford assay to see protein concentration, and we recognised that it could not provide quantitative data on molecular weight of proteins.

We chose to use the Ni-NTA to purify the proteins as it would yield a pure sample that can be further tested on and is within budget. To accommodate for this procedure, we included a 6-His tag in the protein that will bind to the Ni ions.

Tips and tricks:

  • With SDS-pages, make sure there is enough running buffer as it had short-circuited the machine
  • Be careful when loading the protein samples as if done too quickly, it may splash on to surrounding wells, obscuring results.
  • The SDS-pages need to be destained until most of the gel is clear to ensure best photo clarity when analysing.
  • When heating water baths up to 95ºC, it is more efficient to boil water and pour it into the water bath
  • Pay attention to any guides provided by the manufacturer, especially the units of proteases
  • If it is not possible to destain a gel immediately using the destaining solution, leave the gel in distilled water to slowly destain overnight
  • The density of the sample should be greater than the density of the solution in the well

Part 1: Transformation of PET21a+balcp19k plasmids into BL21(DE3) competent cells

Agar Plate Results
  1. Transformation of Recombinant Plasmid into Expression Host
  2. Take competent E. coli BL21(qDE3) cells from a -70 °C freezer and thaw them on ice.
  3. Add 1 μL of pET21a-balcp19k, to 50 μL of competent E. coli BL21(DE3) cells. Incubate on ice for 30 minutes.
  4. Heat shock at 42 °C for 60 seconds, then return to ice for 5 minutes.
  5. Add 950 μL of antibiotic-free LB liquid medium (or SOC) to the mixture and shake at 220rpm/min at 37ºC for 1 hour.
  6. Centrifuge the cultured cells at 6800g for 3 minutes. Discard 800ul of the supernatant using a pipette tip and resuspend the cells in the remaining supernatant.
  7. Plate 100 μL of the resuspended mixture onto LB agar plates containing ampicillin. After the liquid is fully absorbed, invert the plates and incubate at 37 °C overnight.

Part 2: Inoculating a Liquid Bacterial Culture

Inoculation Setup
Culture Growth
  1. Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle:4 g NaCl, 4 g Tryptone, 2 g Yeast Extract, and dH2O to 400 mL

Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.

  1. When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration.

[add exp table here]

Note: If you intend to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2L Erlenmeyer flask.

  1. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
  2. Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
  3. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
  4. Incubate bacterial culture at 37°C 250rpm for 12-18 hr in a shaking incubator

(From: https://www.addgene.org/protocols/inoculate-bacterial-culture)

Part 3: Induced Expression of the Cp19k Protein

  1. The next day, dilute the overnight culture 1:100 into 5 mL of fresh LB medium containing ampicillin. Incubate at 37 °C, shaking at 220 rpm/min for 1.5 hours. (Measure OD at 600 nm. When OD₆₀₀ reaches between 0.4–0.6,) add IPTG (1.0mM) to a final concentration of 1.0 mM to induce recombinant protein expression.
  2. After induction at 27 °C for 4 hours, take 1.5 mL of culture. Centrifuge at 10,000 rpm/min for 5 minutes. Discard the supernatant and resuspend the pellet in 400 μL PBS.

Part 4: Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids

  1. After you have bacterial growth, add 600 μL of the overnight culture to 400 μL of 50% glycerol in a 1.5ml centrifuge tube.
  2. Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Avoid freeze and thaw cycles as it reduces shelf life.
  3. To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.
  4. Grow your bacteria overnight at the appropriate temperature. Growth conditions, including copy number and growth temperature, can be found on your plasmid's information page. The next day you will be able to start an overnight culture for plasmid DNA prep the following day.

(From: https://www.addgene.org/protocols/create-glycerol-stock)

Part 5: Extraction and purification of proteins via Ni-NTA column (Purification of 6xHis-tagged Proteins Under Native Conditions)

Ni-NTA Column Purification

Before use, native Lysis Buffer must be supplemented with lysozyme and BenzonaseR Nuclease. Dissolve the contents of the lysozyme vial in 600 μl of native Lysis Buffer. Add 100 μl of the lysozyme solution to a 10 ml aliquot of native Lysis Buffer. Store the remaining lysozyme solution at -20°C. Thaw the vial containing Benzonase® Nuclease solution and add 10 μl to the 10 ml aliquot of native Lysis Buffer.

Materials:

  • E. coli cell pellet from 250 ml culture
  • 2x SDS-PAGE sample buffer

Procedure

  1. Thaw the cell pellet for 15 min on ice and resuspend the cells in 10 ml native Lysis Buffer. Ensure that lysozyme and Benzonase® Nuclease have been added to the lysis buffer.
  2. Incubate on ice for 30 min. Mix 2-3 times by gently swirling the cell suspension.
  3. Centrifuge lysate at 14,000 x g for 30 min at 4°C to pellet the cellular debris. Retain the cell lysate supernatant. The supernatant contains the soluble fraction of the recombinant protein.
  4. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of the supernatant and store at -20°C for SDS-PAGE analysis.
  5. Gently resuspend the resin in a Fast Start Column by inverting it several times.
  6. Break the seal at the outlet of the column, open the screw cap, and allow the storage buffer to drain out. It is important that the outlet seal is broken before the screw cap is removed.
  7. Apply the cell lysate supernatant from step 3 to the column.
  8. Collect the flow-through fraction. Add 5 μl 2 x SDS-PAGE sample buffer to a 5 μl aliquot of the flow-through fraction and store at -20°C for SDS-PAGE analysis.
  9. Wash the column 2 times with 4 ml of native Wash Buffer. Collect both wash fractions. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of each wash fraction and store at -20°C for SDS-PAGE analysis.
  10. Elute bound 6xHis-tagged protein with two 1 ml aliquots of Native Elution Buffer.
  11. Collect each elution fraction in a separate tube. Add 5 μl 2x SDS- PAGE sample buffer to a 5 μl aliquot of each elution fraction and store at -20°C for SDS-PAGE analysis.
  12. Analyze all fractions by SDS-PAGE.

- approx. 25mg/L yield

(From: EN-QIAexpress-Ni-NTA-Fast-Start-Handbook)

Part 6: Protein Digest for native proteins

  1. Reconstitute lyophilized powder in suitable/recommended solvent to form a concentration of 50mM. For Trypsin, use acetic acid, for alcalase, use Tris HCl with pH range between 7-9. Adjust volume of reconstituted solution to account for protease:protein (unit/mg) ratio.
  2. Incubate at 37ºC from 4 hours to overnight
  3. Take an aliquot and add SDS-page 4x loading buffer
  4. Heat shock for 10 minutes at 95ºC

(Adapted from https://www.upstate.edu/proteomics/prep/trypsin.php)

Part 7: SDS-PAGE to analyse the results of enzyme digest

SDS-PAGE Setup
SDS-PAGE Results

Materials:

  • SDS-PAGE gel (e.g., 12% resolving, 4% stacking)
  • Protein samples
  • 4x Loading buffer
  • 95 ºC water bath
  • Protein ladder (molecular weight marker)
  • Electrophoresis tank and power supply
  • Running buffer (1× Tris-Glycine-SDS)
  • Micropipettes and tips

1. Prepare Samples

  1. Mix protein sample (e.g., lysate, supernatant, pellet) with SDS-PAGE loading buffer in the ratio of 3:1
  2. Heat all samples at 95°C for 5 minutes to denature proteins and ensure uniform charge.
  3. Briefly spin down to collect condensation.

2. Set Up Gel

Insert SDS-PAGE gel into electrophoresis tank.

Fill inner and outer chambers with 1× running buffer (3:1, water: buffer).

3. Load Samples

  1. Load 40μL of protein ladder in the first and/or last lane.
  2. Load 40μL of sample (with loading buffer) per lane (depending on well size).

4. Run the Gel

  1. Connect electrodes: black (–) to top, red (+) to bottom.
  2. Run at 120 V through the resolving gel until the dye front is near the bottom, make sure to not let the dye front run through. (~45–60 minutes).

5. Post-Electrophoresis

Carefully open the seal on the inner chamber of the SDS-page machine

Remove the gel (sandwiched in between two glass plates)

Use a spatula to carefully pry open the glass and remove the gel

Place the gel in

Rinse the gel with distilled water twice, carefully pouring out the water afterwards

Add Coomassie Brilliant Blue stain until it just covers the gel.

Shake for 1-2 hours

Pour out the stain and recycle it

Add destaining solution until it just covers the gel

Shake for 30-90 mins

Repeat previous 2 steps with new destaining solution until the bands are visible (leave overnight for better effects).

Notes

Use fresh gels for clearer resolution.

12% resolving gel is suitable for proteins ~20–100 kDa.

Keep your protein ladder visible to estimate molecular weights. It is recommended to add the standard to the 1st and last well of the gel to account for uneven dye fronts.

Run multiple sample types (lysate, supernatant, pellet) to check protein solubility.