Overview of the E. coli Expression System

The E. coli expression system is one of the earliest and most established platforms in recombinant protein production. It remains widely used in molecular biology and biotechnology due to its well-characterized genetic background, rapid growth rate, and ability to achieve high-level protein expression.

Key advantages of the E. coli system include:

• High protein yield and scalability
• Short culture and expression cycles
• Cost-effective cultivation
• Strong resistance to contamination
• Extensive availability of expression vectors and host strains

These features make E. coli an ideal host for both research-scale and industrial protein production.

Challenges in Protein Purification

While advances in genetic engineering have significantly simplified protein expression, protein purification remains a critical bottleneck. The purification process is often time-consuming, technically demanding, and highly dependent on protein properties such as solubility, stability, and folding.

To overcome purification challenges,The most commonly used tag is the polyhistidine tag (His-Tag) due to its:

• Small size and minimal impact on protein structure
• Strong affinity for metal ions
• Compatibility with various expression systems

Proteins containing a His-tag can be efficiently purified using Immobilized Metal Ion Affinity Chromatography (IMAC).Regardless of whether the recombinant protein is expressed in a soluble form or as inclusion bodies, IMAC provides a reliable and efficient purification strategy.

Experimental Steps

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Protein Expression and Sample Processing (tMAT)

1. Pick a single colony from the transformed BL21 plate and inoculate it into 5 mL LB liquid medium (containing 0.1% antibiotic). Incubate at 37°C with 220 r/min shaking for 8 h.

2. Expand the culture by inoculating the 5 mL seed culture at a 1:100 ratio into 500 mL LB liquid medium (containing 0.1% antibiotic). Culture at 37°C until OD₆₀₀ = 0.8–1.0.

3. Cool down for adaptation by adjusting the shaker temperature to 30°C and continuing incubation for 0.5 h.

4. Take the pre-induction sample (NI) by transferring 0.5 mL of bacterial culture into a microcentrifuge tube. Centrifuge at 4°C, 12,000 rpm for 2 min, discard the supernatant, and keep the pellet for later use.

5. Add IPTG for induction to a final concentration of 0.5 mmol/L to the remaining bacterial culture.

6. Perform induction by incubating at 30°C with 220 r/min shaking for 6 h.

7. Take the post-induction sample (I) by transferring 0.5 mL of bacterial culture into a microcentrifuge tube. Centrifuge at 4°C, 12,000 rpm for 2 min, discard the supernatant, and keep the pellet for later use.

8. Harvest the cells by centrifuging the remaining bacterial culture at 4,000 rpm for 20 min, discard the supernatant, and collect the cell pellet.

9. Resuspend the cell pellet thoroughly in 50 mL of lysis buffer and place on ice.

10. Perform sonication with the following settings: 3 s on / 9 s off, power 30%–40%, alarm temperature 25°C (use an ice bath). Continue until the bacterial suspension becomes clear.

11. Separate the supernatant and pellet by centrifuging the sonicated lysate at 13,000 rpm, 4°C for 20 min. Carefully transfer the supernatant to a new tube.

12. Prepare the S sample by mixing 30 μL of supernatant + 5 μL of 4× protein loading buffer. Heat at 100°C in a metal bath for 5 min.

13. Prepare the P sample by transferring a small piece of pellet into a microcentrifuge tube, adding 80 μL of 1× protein loading buffer, mixing well, and heating at 100°C in a metal bath for 5 min.

Affinity Chromatography Purification (Ni-NTA)

1. Prepare the affinity column: Secure the Ni-NTA affinity column on a stand. Remove the bottom cap and allow the storage solution to flow through. Place a clean beaker under the column.

2. Equilibrate the affinity column: Equilibrate the column with 5–10 column volumes of Ni-lysis buffer. Control the flow rate so that the liquid level does not drop below the top of the resin.

3. Load the sample: Slowly add the supernatant (remaining part of the S sample) obtained after high-speed centrifugation onto the affinity column. Collect the flow-through in the beaker below.

4. Collect the flow-through sample (F): After all the supernatant has passed through the column, slowly add the flow-through from the beaker back onto the column again. Take 30 μL of the solution from the beaker + 5 μL of 4× protein loading buffer, heat at 100°C in a metal bath for 5 min, and label as F sample.

5. First wash (W1): Wash the column with Ni-lysis buffer, adding 5–10 mL each time, until the Coomassie Brilliant Blue detection solution remains blue-free. Take 30 μL of the first two drops of flow-through + 5 μL of 4× protein loading buffer, heat at 100°C in a metal bath for 5 min, and label as W1 sample.

6. Second wash (W2): Continue washing with Ni-lysis buffer until the Coomassie Brilliant Blue detection solution remains blue-free (do not collect the flow-through at this stage). Take 30 μL of the first two drops of flow-through + 5 μL of 4× protein loading buffer, heat at 100°C in a metal bath for 5 min, and label as W2 sample.

7. Wash control (BAW): Take 30 μL of the first two drops of flow-through + 5 μL of 4× protein loading buffer, heat at 100°C in a metal bath for 5 min, and label as BAW sample.

8. Elution (E): Elute the target protein using Ni-elution buffer, adding 1 mL each time and collecting the flow-through. Repeat until the Coomassie Brilliant Blue detection solution remains blue-free (indicating no more protein elution). Take 30 μL of the first two drops of flow-through + 5 μL of 4× protein loading buffer, heat at 100°C in a metal bath for 5 min, and label as E sample.

9. Resin control (BAE): After the column has drained completely, use a pipette tip to transfer 15 μL of the affinity column resin into a microcentrifuge tube. Add an appropriate amount of protein loading buffer, heat at 100°C in a metal bath for 5 min, and label as BAE sample.

10. Sample storage: After all samples have been heated (the proteins are now denatured), they can be stored at room temperature. It is recommended to proceed with SDS-PAGE electrophoresis as soon as possible.