Allele Biotech’s custom protein expression group has expressed hundreds of proteins in various species of bacteria in the past 10 years. One of the major reasons that can potentially hinder expressing mammalian proteins in E. coli is codon bias. With the advancement in gene synthesis technologies, traditional cDNA cloning methods can be replaced by complete gene synthesis with optimized codons as we often do for our customers. When dealing with protein insolubility problems, we typically test different strains of E. coli, different induction methods, purification buffers and schemes and work with the customer to solve the problems at necessary steps. For instance, we use His-tag for affinity purification if urea is needed to solubilize the protein from bacterial pellets. Through collaboration, we can help our customers with their needs for crystallization.
Phase I: Subcloning into Expression Vector
Phase II: Generation and Identification of Expression Positive Clones
Phase III: Expression Optimization
Phase V: Protein Extraction & Purification
Depending on your research needs, you may choose any starting phase or combinations.
Phase I: Subcloning into Expression Vector
Cat# ABP-CS-PEBT001
Description:
- Amplification/isolation of the gene of interest out of a customer-supplied vector and subcloning it into a T7 promoter based high-level expression vector.
- Verification of the subcloned gene by restriction digest and sequencing.
- Preparation of the recombinant vector DNA.
Required Materials: 5 ug of plasmid containing gene of interest plus complete vector sequence in electronic format.
Deliverables:
- 20 ug of expression-ready recombinant pET plasmid containing the gene of interest
- Final sequencing data
- Detailed research report
Duration: 2 weeks; If insert is > 2kb, an additional week per 1kb.
Price (USD): $999; If insert is > 2kb, an additional $399 per 1kb.
Optional: BL21(DE3) expression ready plates are also available, with a price of $29 per plate.
Phase II: Generation and Identification of Expression Positive Clones
Cat# ABP-CS-PEBT002
Description:
- Transformation of recombinant constructs into a suitable E .coli strain.
- Small scale induction to over-express target protein with common or customer-provided protocols. For further optimization, you may choose our Expression Optimization.
- Test for recombinant protein by SDS-PAGE and Western Blot (customer must provide appropriate antibodies).
Required Materials: 5 ug of expression-ready recombinant pET plasmid containing the gene of interest plus complete vector sequence in electronic format.
Deliverables:
- Expression positive clone (E. coli glycerol stock or Plate)
- SDS-PAGE and Western Blot data
- Detailed research report
Duration: 1 week
Price (USD): $299
Optional: For His or GST tagged protein, you may choose to use our antibodies in stock, with an additional charge of $29.
Phase III: Expression Optimization
Cat# ABP-CS-PEBT003
Description:
- Culture broths, inducer concentration, induction starting points, induction time, induction temperature, optimized lysis buffer for better yield and solubility, will all be tested in detail.
- If necessary, certain special optimizations will be suggested by our specialists.
- Test for recombinant protein by SDS-PAGE and Western Blot (customer must provide appropriate antibodies).
Required Materials: 5 ug of expression-ready recombinant pET plasmid containing the gene of interest plus complete vector sequence in electronic format.
Deliverables:
- Expression positive clone (E. coli glycerol stock or Plate)
- SDS-PAGE and Western Blot data
- Detailed research report
Duration: 2 weeks
Price(USD): $599
Optional: For His or GST tagged protein, you may choose to use our antibodies in stock, with an additional charge of $29.
Phase IV: Large-scale Culture
Cat# ABP-CS-PEBT004
Description:
- One liter of bacterial culture will be induced with an appropriate inducer (e.g., IPTG, IAA) and harvested for protein purification.
- Test for recombinant protein by SDS-PAGE and Western Blot (customer must provide appropriate antibodies).
Required Materials: Expression positive clone (E. coli glycerol stock or plate) together with a verified induction procedure.
Deliverables:
- Cell pellets from 1 liter fermentation
- SDS-PAGE or Western Blot data
- Detailed research report
Duration: 2 days
Price (USD): $199
Optional:
- For His or GST tagged protein, you can choose to use our antibodies in stock, with an additional charge of $29.
- You can provide expression ready plasmids, or for an addtional $29, clones may be prepared.
- Inquire for volume discount if > 10 liters of culture.
Phase V: Protein Extraction & Purification
Cat# ABP-CS-PEBT005
Description:
- Cell pellet harvested from 1 liter of bacterial culture. After induction, cells will be lysed with our optimized reagents and protocol.
- Protein purification using appropriate affinity resin for 6xHis or GST-tagged recombinant proteins.
- Purification can be performed either under native or denatured conditions (as desired by the customer).
- SDS Page and Western Blot will be performed for quality testing.
Required Materials: Cell pellet harvested from 1 liter of bacterial culture after induction.
Deliverables:
- Purified protein in standard or customer-specified solution
- SDS-PAGE or Western Blot data
- Detailed research report
Duration: 2-3 weeks
Price (USD): $1500/L, discounts offered for larger volumes.