Dear colleagues
Please suggest a comprehensive and complete protocol for the production and purification of bacterial recombinant protein in E.Coli BL21 strain.
The following is a generalized protocol for the production and purification of a recombinant protein using the E. coli BL21 strain. Please note, the details may vary based on the specific properties of your target protein and plasmid construct.
1. Transformation:
a. Thaw a tube of competent E. coli BL21 cells on ice.
b. Add 1-10 ng of your plasmid DNA to the tube.
c. Incubate the cells on ice for 30 minutes.
d. Heat-shock the cells by placing them in a 42°C water bath for exactly 30 seconds.
e. Immediately transfer the cells back to ice and incubate for another 2 minutes.
f. Add ~200-900 μL of room temperature SOC media to the cells.
g. Shake the tube gently at 37°C for 1 hour.
h. Spread 50-100 μL of the cells onto a pre-warmed selective agar plate.
i. Incubate the plate at 37°C overnight (~16 hours).
2. Growing the Transformants and Inducing Protein Expression:
a. Pick a single colony from the agar plate and inoculate a pre-warmed tube of LB medium containing the appropriate antibiotic.
b. Incubate the culture at 37°C with shaking until it reaches mid-log phase (OD600 ~ 0.6-0.8).
c. Induce protein expression by adding an inducer (like IPTG) to a final concentration of 0.5-1.0 mM.
d. Continue to incubate the culture for 3-4 hours at 37°C with shaking.
3. Cell Harvesting:
a. Centrifuge the culture at ~5000 g for 10-15 minutes at 4°C to pellet the cells.
b. Discard the supernatant and resuspend the pellet in an appropriate volume of lysis buffer.
4. Cell Lysis:
a. Lyse the cells using your preferred method (e.g., sonication, chemical lysis, or freeze-thaw cycles).
b. Centrifuge the lysate at ~15,000 g for 20-30 minutes at 4°C to pellet the cellular debris.
c. Carefully collect the supernatant, which contains the soluble fraction of your protein.
5. Protein Purification:
a. Prepare a column with your affinity resin of choice, equilibrated with binding buffer.
b. Load the supernatant onto the column and allow the protein to bind to the resin.
c. Wash the column with wash buffer to remove non-specifically bound proteins.
d. Elute the target protein using an elution buffer.
6. Protein Dialysis and Concentration:
a. Dialyze the protein against a suitable buffer to remove the elution buffer and exchange into a buffer suitable for your downstream applications.
b. Concentrate the protein using a centrifugal filter unit, if necessary.
7. Protein Analysis:
a. Analyze the purity of your protein using SDS-PAGE.
b. Confirm the identity of your protein using Western blot or mass spectrometry.
Remember to keep everything sterile and work in clean conditions to avoid any contamination. Additionally, the use of controls is important to ensure your procedure worked as expected. This is a general protocol, and may need to be optimized depending on the specific protein of interest. It's also recommended to follow specific protocols associated with the plasmid, strain, or purification kit being used.
Download the pET system manual
https://www.merckmillipore.com/INTL/en/search/pET%20system%20manual?search=&TrackingSearchType=SB+-+Search+Box&SearchContextPageletUUID=&SearchTerm=pET+system+manual
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