Experimental Protocol for Prokaryotic Protein Expression

Determine the gene sequence

Based on the experimental requirements, locate the target gene sequence on NCBI.

Select an Expression Vector

Choose an appropriate vector; common prokaryotic expression vectors include the pET series, pGEX-4T-1, pET-SUMO, and others.

Select the host to express

BL21 (DE3), Rosetta series strains

Construction of a Prokaryotic Expression Plasmid

Select appropriate restriction sites using MCS. After performing a dual restriction digestion on both the vector and the target gene, proceed with gel recovery, ligation, transformation, and sequencing verification.

Expression and Purification of Recombinant Proteins

First, the plasmids were transformed into DH5α and BL21 competent cells, respectively. Single colonies were then selected, and the bacterial cultures were sent for DNA sequencing. After sequencing, the results were aligned using Snapgene software. The alignment results are provided in the appendix. The plasmids were promptly purified on a small scale for cloning into a plasmid library and storage, while the bacterial cultures were also preserved.

Expression and Purification of Recombinant Proteins

After transformation, inoculate the culture with 1% of the bacterial suspension and add kanamycin at a concentration of 0.1%. Incubate at 37°C and 220 r/min. After 6 hours, measure the OD600 nm value; the optimal range is 0.6 to 0.8.

Expression and Purification of Recombinant Proteins

Once the bacterial culture reaches the logarithmic growth phase, add IPTG at a concentration gradient determined in preliminary experiments to achieve a final concentration of 1.0 mmol/L. Incubate at 37°C and 220 r/min for 6 hours, then centrifuge at 8000 r/min for 3 minutes, collect the bacterial pellet, and wash the pellet three times with PBS.

Cell Lysis

Next, the bacterial pellet must undergo ultrasonic disruption and lysis at room temperature. Resuspend the pellet from 1 L of bacterial culture in 20 mL of PBS to disrupt the cells. After sonicating at 10 W for 1 hour, centrifuge at 12,000 rpm for 15 minutes to collect the cells. Remove the supernatant, resuspend the pellet in lysis buffer, and lyse at room temperature for 2 hours. After lysis, centrifuge at 12,000 rpm for 15 minutes, retain the supernatant to obtain the protein lysate, and filter it through a 0.45-μm filter.

Polyacrylamide Gel Electrophoresis

After protein expression, the expression level is assessed via polyacrylamide gel electrophoresis. The expression of the target band before and after induction is compared to confirm whether expression was successful.

Purification of the Target Protein

(Select the appropriate purification method based on the type of tag.)

At this point, the protein lysate contains all proteins. To purify the target protein, a Ni-NTA pre-packed gravity column was selected, taking advantage of the binding affinity between nickel ions and the His tag. The specific purification procedure is described as follows: Drain the pre-packed buffer, wash the column with double-distilled water, add 10 mL of binding buffer (8 M urea PBS containing 5 mM imidazole) to equilibrate the column, set the rotation speed to 25 r/min, and load the protein; repeat this step three times; Next, wash out contaminants with Binding Buffer until the Protein A 280 absorbance approaches the baseline; then switch to Elution Buffer (8 M urea PBS containing 500 mM imidazole) to elute the target protein, collect the sample, and record its concentration; After washing until the Protein A 280 absorbance of the solution approaches the baseline, condition the column, then wash with 5 times the column volume of Binding Buffer until the solution’s absorbance returns to the baseline. Wash with double-distilled water, and finally store the protein in 20% ethanol. Note: Do not allow the column to run dry at any point during the procedure, as this may damage the column media and affect purification efficiency.

Determine protein concentration and verify