E. coli Expression Experimental Protocol

Recombinant protein expression in Escherichia coli (E. coli) is widely used in molecular biology and biotechnology due to its ease, cost-effectiveness, and rapid growth. This E. coli expression protocol outlines a standardized workflow for cloning, transformation, protein expression, and purification in the E. coli system, ensuring reproducibility and efficiency.

Materials And Equipment

Strain: BL21(DE3), DH5α, or other suitable E. coli expression host.
Plasmid: Expression vector (e.g., pET, pGEX, pMAL).

Cat.No.	Product Name	Price
VET1026	pGEX-5X-1 vector	Inquiry
VET1027	pGEX-6P-1 vector	Inquiry
VET1028	pGEX-6P-2 vector	Inquiry
VET1029	pGEX-2TK vector	Inquiry
VET1030	pBV220 vector	Inquiry
VET1031	pBV221 vector	Inquiry
VET1032	pBV222 vector	Inquiry
VET1033	pTrcHisA vector	Inquiry
VET1034	pTrcHisB vector	Inquiry
VET1035	pTrcHisC vector	Inquiry
VET1036	pBAD24 vector	Inquiry
VET1037	pBAD43 vector	Inquiry
VET1038	pBAD/His A vector	Inquiry
VET1039	pBAD/His B vector	Inquiry
VET1040	pBAD/His C vector	Inquiry

Media: LB broth, Terrific Broth (TB), or autoinduction medium.
Antibiotics: Ampicillin, kanamycin, or chloramphenicol (depending on the plasmid).
Inducer: Isopropyl-β-D-1-thiogalactopyranoside (IPTG) or lactose.
Buffers: Lysis buffer, bind/wash/elute buffer for purification.
Equipment: shaking incubator, centrifuge, ultrasonic disruptor, SDS-PAGE electrophoresis apparatus, chromatography system (e.g. AKTA).

Experimental Procedures

1. Cloning and plasmid preparation

(1) Gene insertion cloning: Amplify the target gene by PCR and ligate into an E. coli expression vector.

(2) Transformation: Introduce the plasmid into competent E. coli cells (e.g., DH5α for cloning, BL21(DE3) for expression) by heat shock or electroporation.

(3) Colony screening: Screen positive colonies by antibiotic resistance and confirm by colony PCR or sequencing.

2. Small-scale expression test

(1) Inoculation: Pick a single colony and add it to 5 mL LB+antibiotic medium, and culture it at 37℃, 200 rpm overnight.

(2) Main culture: Dilute the colony in fresh medium (LB/TB) at a ratio of 1:100 and culture to OD600 of about 0.6-0.8.

(3) Induction: Add IPTG (0.1-1 mM) or lactose, and incubate at 16-37℃ (4-24 hours, depending on solubility requirements).

(4) Harvest: Pellet cells by centrifugation (4,000 X g, 10 min, 4℃).

3. Protein extraction and analysis

(1) Lysis: Resuspend the pellet in lysis buffer and lyse by sonication or enzymatic methods (e.g., lysozyme).

(2) Centrifugation: Clarify lysate (12,000Xg, 30 min, 4℃) to separate soluble/insoluble fractions.

(3) SDS-PAGE: Analyze supernatant and pellet fractions to confirm expression and solubility.

4. Large-scale purification

(1) Affinity chromatography: Purify His-tagged proteins using Ni-NTA resin under native/denaturing conditions.

(2) Buffer exchange: Dialyze or desalt eluted protein into storage buffer (e.g., PBS, Tris-HCl).

(3) Concentration: If necessary, concentrate protein using centrifugal filters (e.g., Amicon).

5. Quality control

(1) Purity: Assessed by SDS-PAGE/Coomassie blue staining.

(2) Concentration: Measure by Bradford/BCA assay or spectrophotometry (A280).

(3) Functionality: Verify by activity assay (e.g., enzymatic assay, Western blot).

Troubleshooting

Low expression: Optimize induction conditions (temperature, IPTG concentration, induction time).
Insolubility: Test lower induction temperature (16-25℃) or solubility tags (e.g., GST, MBP).
Degradation: Add protease inhibitors or use a protease-deficient strain (e.g., BL21(DE3)pLysS).

* For research use only. Not intended for any clinical use.

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