info@mail.ucallm.com
1. Overview
The Escherichia coli (E.coli) protein expression system is the most widely used prokaryotic expression platform for recombinant protein production. As an essential tool in molecular biology research and biotechnological industrialization, this system offers clear genetic background, high-level target gene expression, short culture cycles, and strong contamination resistance. The system typically utilizes plasmid vectors carrying the gene of interest, leveraging the host's efficient transcription and translation machinery to achieve high-level recombinant protein production.
After decades of research and development, E. coli has become one of the preferred hosts for recombinant protein expression, with recombinant proteins accounting for up to 50% of the total cellular protein under optimized conditions. The system is widely applied in basic research, industrial enzyme production, diagnostic reagent development, and therapeutic protein manufacturing.
2. Core Advantages
(1) Well-characterized genetic background: One of the best-studied model organisms, facilitating genetic manipulation and expression optimization
(2) Rapid growth and short cycle: ~20 minutes doubling time enables large quantities of recombinant protein in a short period
(3) High expression levels: For some proteins under optimized fermentation conditions, yields can reach several grams per liter.
(4) Low cost: Simple culture conditions requiring only basic nutrients and standard equipment
(5) Ease of manipulation: High transformation efficiency and extensive molecular biology toolkits
(6) Scalability: Easily scalable from small laboratory cultures to large-scale industrial fermentation
3. System Limitations
(1) Lack of native PTM machinery: Lacks most eukaryotic post-translational modifications (PTMs), including glycosylation and many phosphorylation events.
(2) Limited disulfide bond formation in the cytoplasm: The reducing cytoplasmic environment is not conducive to proper disulfide bond formation(requires specialized strains or periplasmic translocation)
(3) Inclusion body formation: Overexpression often leads to insoluble protein aggregates (inclusion bodies)
(4) Codon bias: The codon bias of E. coli may affect the expression of eukaryotic genes
(5) Protein degradation: Exogenous proteins are susceptible to host proteases
(6) Limited extracellular secretion: Has limited protein secretion capability; recombinant proteins are typically accumulated in the cytoplasm or periplasm.
(7) Endotoxin risk: Lipopolysaccharide (endotoxin) in the cell wall may contaminate final products
4. Key Application Areas
(1) Basic research: Production of recombinant proteins for structural/functional studies, e.g., histones for chromatin remodeling studies, luciferase for bioluminescence imaging.
(2) Industrial enzyme production: Proteases, lipases, amylases, etc., e.g., phytase for animal feed additives, chymosin for cheese production.
(3) Therapeutic proteins: Insulin, human growth hormone (hGH), etc., e.g., recombinant human insulin and interferons.
(4) Diagnostic Reagents: Antigens, antibodies, and enzyme labels, e.g., C-reactive protein antigen for CRP testing.
(5) Vaccine development: Recombinant subunit vaccines, e.g., hepatitis B surface antigen (HBsAg) for hepatitis B prevention
5. Classification of Expression Vectors
5.1 By promoter type
Type | Promoter | Features | Vectors |
IPTG-inducible | lac, tac, trc, T5/lac operator | Widely used; induced by IPTG; tight regulation | pET series, pQE series |
Heat-inducible | λPL, cspA | Induced by temperature shift (e.g., 42℃) | pEX series |
T7 system | T7 bacteriophage promoter | One of the strongest; requires host-provided T7 RNA polymerase | pET series |
Cold‑shock | E. coli cold‑shock protein A (cspA) promoter | Low‑temperature induction (typically 15-20℃); suppresses host protein background; markedly improves target solubility | pCold series |
5.2 By expression product format
Type | Features | Representative Vectors |
Non-fusion Protein Vectors | Native protein without fusion tags | pET series (basic) |
Fusion Protein Vectors | N- or C-terminal affinity tags for purification and solubility enhancement | pET series (His-tagged), pGEX series (GST-tagged), pMAL series (MBP-tagged) |
Secretory Vectors | Signal peptide for periplasmic or extracellular secretion | pEZZ18, etc. |
5.3 Common Fusion Tags
Tag | Features | Application |
His-tag | Small size (~0.8 kDa); purified by metal chelate affinity chromatography | Most widely used purification tag; often does not require removal |
GST-tag | 26 kDa; promotes correct folding and solubility | Proteins prone to inclusion body formation; requires cleavage |
MBP-tag | Significantly enhances solubility of fusion proteins | Expression of poorly soluble proteins |
Strep-tag II | High affinity for Strep-Tactin | High-purity purification |
Trigger Factor (TF) | Prokaryotic ribosome‑associated chaperone (48 kDa); promotes co‑translational folding | Used with cold‑shock promoters (e.g., pCold TF) for efficient soluble expression |
SUMO tag | Small ubiquitin‑like modifier; promotes correct folding and stability | Used in pCold‑SUMO vectors; enhances solubility of difficult proteins |
Protein S tag | Small highly soluble tag derived from Myxococcus xanthus Protein S | Enhances solubility and stability of fusion proteins |
6. Common Expression Host Strains
Strain | Features | Applications |
BL21 | lon and ompT protease-deficient | Classic widely used expression host; reduces protein degradation |
BL21(DE3) | Has DE3 Iysogen that expresses T7 RNA polymerase; Deficient in lon and ompT proteases; Induced by IPTG | Suitable for routine high-level expression of recombinant proteins |
BL21(DE3) pLys | Expresses T7 lysozyme to suppress basal T7 RNA polymerase activity before induction | Prevents leaky expression ; Suitable for the expression of toxic genes |
Lemo21(DE3) | Contains features of BL21(DE3); Tunable expression of difficult clones by varying the level of lysozyme (lysY) | Suitable for the expression of challenging protein including: toxic proteins, membrane proteins, and low soluble ones |
Tuner (DE3) | Has lac permease (lac Y) mutation | Allows uniform entry of IPTG into all cells in the population; Suitable for toxic and insoluble proteins |
Shuffle | Expresses disulfide bond isomerase DsbC; Deficient in proteases Lon and Omp | Promotes cytoplasmic disulfide bond formation by expressing DsbC |
Rosetta | Supplies tRNAs for rare codons | Expression of eukaryotic genes containing rare E. coli codons |
Origami | Has mutation in trxB and gor genes | Expression of proteins requiring disulfide bonds |
C41(DE3) and C43(DE3) | Improved tolerance to toxic protein expression | Suitable for the expression of toxic and/or membrane proteins form all classes of organisms |
DH5α | recA- and endA- | Plasmid construction and amplification |
7. Selection Guide
Requirement | Recommended Vector/System | Reason |
Routine high-level expression | pET series + BL21(DE3) | T7 promoter; high expression levels; well-established |
His-tag purification | pET series (His-tagged) | One-step affinity purification; small tag often doesn't need removal |
Solubility enhancement | pGEX / pMAL series | Promotes correct folding; reduces inclusion bodies |
Difficult eukaryotic genes | Rosetta series + conventional vector | Supplies rare codon tRNAs |
Secretory expression | pEZZ18 | Signal peptide-mediated secretion into the periplasm |
Selection Recommendations: BL21(DE3) for routine expression; Rosetta for eukaryotic genes with rare codons; Origami for proteins requiring disulfide bonds; DH5α for plasmid construction and amplification.
8. Service Highlights
l High-Yield Guarantee: Leveraging our extensive E. coli experience, we have successfully achieved gram-scale purified protein production for selected targets
l Multiple Host Options: BL21(DE3), Rosetta, Origami, etc., to meet diverse expression needs.
l Versatile Vector Systems: pET, pGEX, pMAL, etc., supporting fusion tags, non-fusion, and secretion.
l Inclusion Body Refolding: We offer professional refolding services to obtain bioactive soluble protein from inclusion bodies.
9. Service Procedure
Service Step | Gene synthesis and vector construction (Optional) | Expression evaluation and optimization | Protein expression and optimization (Optional) | Large-scale expression and purification |
Description | l Provide us with the protein/gene sequence or the plasmid l Codon optimization l Cloning into expression vectors l Plasmid validation and preparation | l Transformation into E. coli expression host l Pilot expression & purification trial l Protein expression evaluation (SDS-PAGE) | l Soluble protein purification l Refolding & purification of inclusion bodies l QC analysis (SDS-PAGE, UV, etc) | l Large-scale Protein purification l QC analysis
|
Deliverables | l Plasmid sequence l COA report | l Expression evaluation result | l Purified proteins l COA report | l Purified proteins l COA report |
Turnaround Times | 2-3 weeks | 1-2 weeks | 2-3 weeks | 2-3 weeks |
10. Case Studies
11. Frequently Asked Questions (FAQs)
l Q1: My protein always forms inclusion bodies in E. coli. What should I do?
A: Try lowering induction temperature and IPTG concentration, using solubility-enhancing tags, choosing strains like Origami for disulfide bond formation, or performing inclusion body refolding. We offer comprehensive refolding services.
l Q2: How do I choose a fusion tag?
A: For purification only, use His-tag. For solubility enhancement, try GST or MBP. For functional studies, consider the tag's impact on activity and include a protease cleavage site for removal.
l Q3: My protein is toxic to E. coli. What should I do?
A: Use a tightly regulated promoter (e.g., T7 lac promoter in pET series) or specialized strains like Tuner or C41(DE3), and optimize induction conditions.