E. Coli System
The bacterial protein expression system is the most widely applied expression system for producing recombinant proteins. The advantages of E. coli expression system are low cost, high expression level, ease of scale up, and a short turnaround time. However, expression level and solubility are two main bottlenecks for E. coli expression. Our unique codon optimization and parallel screening process can significantly improve protein expression level, while our proprietary refolding methods can significantly increase success rate for protein solublization and yield. With over 500 batches of protein delivered, supported by our experienced services scientists, Pepmic provides services to meet your research needs.

Our Gene-to-Protein service provides guarantees to our clients to deliver a minimum of 4 mg of purified soluble protein. With a minimum set up fee, we will evaluate protein expression and screening for optimized conditions. If the target protein cannot be purified to guaranteed level, no additional charge will be applied.

Phase I: Subcloning into Expression Vector

Phase II: Generation and Identification of Expression Positive Clones

Phase III: Expression Optimization

Phase IV: Large-scale Culture

Phase V: Protein Extraction & Purification

Phase I: Subcloning into Expression Vector

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):

  • $960; If insert is > 2kb, an additional $350 per 1kb

Optional:

  • BL21(DE3) expression ready plates are also available, with a price of $25 per plate

Phase II: Generation and Identification of Expression Positive Clones

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):

  • $260

Optional:

  • For His or GST tagged protein, you may choose to use our antibodies in stock, with an additional charge of $25

Phase III: Expression Optimization

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):

  • $540

Optional:

  • For His or GST tagged protein, you may choose to use our antibodies in stock, with an additional charge of $25

Phase IV: Large-scale Culture

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):

  • $160

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

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):

  • $1350/L, discounts offered for larger volumes