Experiments

1.Remove competent bacteria from -80°C and let thaw in ice (check with gentle flicks)

2. Add 2 µl of plasmid to each of the three types of competent cells and incubate on ice for 15 minutes.

3. Let sit on ice for 30 mins

4. Heat shock the cells at 42°C for 90 seconds

5. Place on ice for 2 mins

6. Add 1 mL of LB medium and place in shaking-culture (37°C, 200rpm) for 45 mins

7. Centrifuge at 4000 rpm for 3 minutes

8. Remove 800mL of clear liquid

9. Centrifuge at 4000 rpm for 3 minutes.

10. Resuspend in remaining LB and add solution to desired container of Kanamycin infused LB

11. Place in shaking-culture (37°C, 200rpm) for 24h

1. Inoculate transformed E.Coli (1mL) into kanamycin LB medium (4mL)

2. Incubate at 37°C with shaking (209 rpm) for 1.5 hours

3. Measurement of optical density (OD) at 595nm

4. When OD=0.6-0.8, induce expression with IPTG (1 M, dissolved in ddH₂O) at 1:1000

5. Incubate at 16°C with shaking (220 rmp) for 20 hours.

1.After expression, centrifugate at 12000rpm for 1 min and remove clear liquid

2.Collect 400 µL of the bacterial culture

3.Resuspend using 80μL lysing buffer (adding 100mmol HEPES, 100mmol NaCl, 1mmol EDTA, 10% glycerinum and 0.5% Triton X-100) per tube

4.Add protease inhibitor

5.Sit on ice for 5-10 mins

6.Add 5XSDS loading buffer (20μL per tube)

7.Heat at 96°C for 10 mins

8.Load upper liquid as samples in SDS-PAGE

1.Preparation of Electrophoresis Tanks: Clean and assemble the electrophoresis cassettes and tanks. Test the seal of the tanks by injecting water and observing the liquid level. Proceed if the liquid level shows minimal to no movement.

2.Preparation of SDS-PAGE Gel: Prepare the separation gel and pour 6mL into each glass plate.Seal the top with anhydrous ethanol. After the gel has polymerized, remove the ethanol and pour in the stacking gel, then insert the comb. Wait for the gel to set.

3.3.Sample Loading and Electrophoresis: Load 5 µl of protein ladder into the first well of each tank. Inject the prepared sample supernatants into the remaining wells. The proteins in the first tank will be stained using Coomassie Brilliant Blue, while the second tank will be used for Western blotting. Load 15 µl of sample into each well of the first tank and 10 µl into the second tank. Additionally, load 8 µg of lactoferrin in the last well of the first tank, and 5 µl in the last well of the second tank. Prepare the SDS-PAGE running buffer and fill the electrophoresis tank. Run the gel at 120V, for 45 mins.

1. Cut the selected gel strip with a surgical blade, place it in the EP tube, record the strip number and corresponding position, and cut the strip into 2 cubic millimeters

2. Add 500μL ultra-pure water (as the liquid level has not exceeded the volume of colloidal particles) and shock for 5 mins each time, and blot dry

3. Add acetonitrile (ACN)/50mM NH₄HCO₃=2:3 solution 500μL, ultrasonic decolorization for 5 mins or shock decolorization for 30 mins, and blot dry

4. Repeat step 3 until the blue fades

5. Add ACN 500μL to dehydrate, shake until the colloidal particles completely turn white, and blot dry (If the colloidal particles do not harden, repeat step 5)

6. Add 10mM dithiothreitol (DTT) (prepared with 10μL MDTT, 990μL 25mM NH₄HCO₃)100ul and bathe in water at 56℃ for 45 mins

7. Blot dry and quickly add 40mM iodoacetamide (IAM) (prepared with: 20μL 1M IAM, 480μL 25mM NH₄HCO₃) 100μL, place in dark for 45mins

8. Wash with 50%ACN solution and ACN successively, dehydrate ACN until the colloidal particles are completely white (standard reference step 5), and blot dry

9. Dilute the 0.1μg/μL enzyme storage liquid 20 times with 25mM NH₄HCO₃. The amount of enzyme liquid added to each EP tube is subject to complete wetting of the colloidal particles, slightly centrifuge, so that the enzyme liquid is fully in contact with the colloidal particles. Place it in 4℃ or on ice for 5 mins, until the solution is fully absorbed by the rubber block, absorb the excess enzyme liquid. Add 25mM NH₄HCO₃ 45μL (immersed in colloidal particles and more than 30μL) in a 37℃ water bath, digest overnight

10. Add 10% formic acid (FA) to terminate the reaction, oscillate and mix, centrifuge, PH

11. Remove the solution and transfer it to a new EP tube, label it, add 60%ACN to the tube with colloidal particles for ultrasonic extraction twice, 10 mins each time, combine the solution three times, vacuum dry the solution, add 0.1%FA aqueous solution 20μL to dissolve the polypeptide

1.Attach the Ni2+ affinity column to the pump head and wash 5 column volumes with a low concentration imidazole salt buffer.

2. Put the protein expressing bacteria solution into a high-speed centrifuge for centrifugation, 5000 rpm, 4 °C, 10 minutes, collect the bacteria, frozen at -20 °C or -80 °C for later use.

3.Resusbend the bacteria with low concentration imidazole buffer.

4.Under the condition of ice bath, release the expressed protein by pressure crusher or ultrasonic crusher. If the bacterial solution is still cloudy after ultrasound, it is necessary to increase the power appropriately and extend the ultrasonic time.

5.Place the suspension in an ultra-high-speed centrifuge at 4°C, 18000 rpm, for 25 minutes, to separate the upper protein albumen and precipitate.

6.SDS-PAGE electrophoresis detection: Determine whether the protein is expressed in soluble or inclusion body (precipitation).

7. Connect the Ni2+ affinity column to the pump head, and the protein cracking solution is passed through the Ni2+ affinity column at a slow and constant speed at 4 °C.

8.Use low concentration imidazole buffer (10 mM imidazole 2-5 mL/min) flow through the Ni2+ affinity column, about 5 column volumes.

9. Perform phase elution with imidazole buffers containing 50, 100, 200, 300 and 400 mM, respectively, with flow rates of 2-5mL/min. Collect eluting peaks at each stage, and each concentration of imidazole buffer flowed through 2 column volumes.

10. Collect protein samples and use SDS-PAGE to detect the molecular weight and purity of the fusion protein.

11.Use ddH₂O to wash 5 column volumes of Ni2+ affinity columns, and then wash 3 column volumes of Ni2+ affinity columns with 20% ethanol at a flow rate of 2-5 mL/min. The columns were always kept in a low temperature environment.

For the culture of Sf9 insect cells, we employed the serum-free and protein-free medium Sf-900™ II SFM. Cells were maintained at 27-28°C in shaking flasks(120-140rmp) until they reached the required cell density (1-2 * 10⁶ cells/mL).

We mixed 50 µL of ExpiFectamine™ Sf reagent with 100 µl of DNA and incubated for 5-10 minutes at room temperature. We then added the DNA-reagent mix to 2 * 10⁶ cells/mL of Sf9 cells and incubated for 5 hours at 27°C.

After the transfection procedure, the cells were cultured at 27°C with shaking for 72 hours. We then harvested the P0 virus stock through centrifugation (1,000 * g for 5 minutes) and stored it at 4°C for immediate use or at -80°C for long-term storage.

The P0 virus stock was inoculated into 50 mL of Sf9 cells (2 * 10⁶ cells/mL) and incubated at 27°C with shaking (120-140 rpm) for 72 hours. We then harvested the P1 virus stock with centrifugation as previously described.

We inoculated Sf9 cells at 2 * 10⁶ cells/mL with the P1 virus stock at a multiplicity of infection (MOI) of 0.01-1 and incubated at 27°C with shaking (120-140 rpm) for 48-72 hours until signs of infection were observed (e.g., enlarged size, reduced viability). We then harvested the cells by centrifugation at 1,000 * g for 5 minutes.

The cell pellets were taken and resuspended in lysis buffer. Cells were lysed with a sonicator and centrifuged at 20,000 *g for 30 minutes.

We used nickel affinity chromatography for purification of the his-tagged recombinant proteins. The column was first loaded with 2 mL of equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0). We then added the supernatant onto the equilibrated Ni-NTA column and incubated for 1 hour at 4°C with gentle mixing to ensure binding between the His-tagged protein and the resin.

To remove non-specifically bound proteins, we washed the column with 10 mL of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0) and discarded the flow-through. To collect the bound target protein, we eluted the column with 2-3 column volumes of elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0). We would then confirm the presence of our recombinant protein through western-blot or mass spectrometry.