Recombinant Protein Production

VectorBuilder offers customized recombinant protein production services employing multiple expression platforms suitable for a wide range of applications including protein structure analysis, enzymatic assay development, drug discovery, diagnostics, and bioengineering. Our protein production process is highly optimized for the generation of high-quality recombinant proteins utilizing our proprietary recombinant protein expression vectors that are also used in our vector cloning services. We can also produce recombinant proteins from customer-supplied vectors.

We offer recombinant protein production based on the following expression platforms:
  • Bacteria
  • Yeast
  • Mammalian cells
  • Insect cells

Service Details

Highlights
  • Comprehensive protein production service with the flexibility to select from a variety of expression platforms to fulfill your research goals.
  • Extensive experience in the optimization and production of various proteins including enzymes, cytokines, receptors, antibodies etc.
  • One-stop solution for the entire protein production workflow starting from vector cloning to protein purification.
  • Options to add a variety of purification or detection tags to meet specific project needs.
  • Availability of multiple purification options to ensure high purity including various chromatography and filtration-based techniques.
  • Post-purification services (e.g., desalting, aliquoting, endotoxin removal, aseptic process and lyophilization) available upon request.
Price and turnaround

Prices and turnarounds of services available for different recombinant protein expression systems are shown in Table 1.

Table 1. Price and turnaround for recombinant protein production.

Recombinant Protein Expression system Available Services Deliverable Typical Yield Price Turnaround
Bacteria (E. coli)** Pilot study*
  • Pilot expression report with SDS-PAGE images
>1 mg,
85% purity
From $300 1-2 weeks
Optimizing protein expression (optional)
  • Optimization data and protocol
From $500 4-6 weeks
Scale-up protein expression and purification (up to 20 L)
  • Purified protein***
  • Detailed protein expression report
From $750 2-3 weeks
Yeast (S. cerevisiae) Pilot study*
  • Pilot expression report with SDS-PAGE images
>1 mg,
85% purity
From $450 1-2 weeks
Optimizing protein expression (optional)
  • Optimization data and protocol
From $600 4-6 weeks
Scale-up protein expression and purification (up to 20 L)
  • Purified protein***
  • Detailed protein expression report
From $900 2-3 weeks
Mammalian cells (293T) Pilot study*
  • Pilot expression report with SDS-PAGE images
1-100 mg/L, 85% purity From $800 3-4 weeks
Optimizing protein expression (optional)
  • Optimization data and protocol
From $1500 3-4 weeks
Scale-up protein expression and purification (up to 1 L)
  • Purified protein***
  • Detailed protein expression report
Please inquire Please inquire
Insect cells (Sf9) Pilot study*
  • Pilot expression report with SDS-PAGE images
1-150 mg/L, 85% purity From $800 6-7 weeks
Optimizing protein expression (optional)
  • Optimization data and protocol
From $1700 6-7 weeks
Scale-up protein expression and purification (up to 1 L)
  • Purified protein***
  • Detailed protein expression report
Please inquire Please inquire

*Please note that the price for a pilot study does not include the cost of cloning the recombinant protein expression vector. You can either send us your own vector or simply send us a design request to have our experts design and clone your vector. Crude extracts from pilot studies can be provided upon request, however they may be prone to degradation during long distance shipping. 

**For expression in bacterial cells, if the recombinant protein is expressed as inclusion bodies additional fees and turnaround may apply for protein recovery.

***Purified proteins are delivered by default in PBS (pH 7.4) containing 50% glycerol, which is suitable for the long-term storage of proteins at -80°C. However, we can deliver proteins in other storage buffers that might be necessary for specific proteins or specific downstream applications upon request.

In addition to the services described above, we offer the following post-purification services described in Table 2.

Table 2. Price and turnaround for additional services.

Available Services Price Turnaround
Western blot* $150/run 2-4 days
Endotoxin testing and removal (< 1.0 EU/mg) $150/sample 5-7 days
Purification tag removal (e.g. His, MBP, GST) $150/sample 5-7 days

* For western blots based on standard fusion tags available in our database, the antibodies will be provided by VectorBuilder. However, for non-standard tags that are not available in our database customers maybe requested to submit their own antibodies.

Customers-supplied materials

If customer-supplied vectors are needed, please submit your materials information on "Support" > "Materials Submission Form". Please strictly follow our guidelines to set up shipment to avoid any delay or damage of the materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur an additional QC charge starting from $120 per item, depending on the type and usage of item. Please note that production cannot be initiated until customer-supplied materials pass QC.

Technical Information

Bacteria

E. coli is the most widely used host for recombinant protein production due to the several advantages it offers including short doubling time, technical simplicity, scalability, and low production costs. VectorBuilder specializes in the production of recombinant proteins in E. coli with a highly optimized workflow for the expression of cytoplasmic, periplasmic as well as soluble and insoluble proteins. In addition, we can generate recombinant proteins in
B. subtilis upon request.

Our typical protein production process starts with construct design including fusion partner selection, purification tag selection, and codon optimization. This is followed by gene synthesis and cloning of the bacterial recombinant protein expression vector. The vector is then transformed into the appropriate bacterial host strain and a small-batch fermentation is performed to evaluate protein expression. Based on expression evaluation we then perform a large-batch fermentation followed by subsequent protein purification.

Typical_workflow_of_recombinant_protein_production_in_E._coli

Figure 1. Typical workflow of recombinant protein production in E. coli.

Yeast

Utilization of yeast as a recombinant protein expression host allows eukaryotic proteins to be produced with many of the post-translational modifications that are essential for their proper function (e.g. glycosylation of therapeutic proteins can reduce the risk of evoking an immune response), while maintaining low production costs compared to other eukaryotic protein expression systems. Other advantages of the yeast expression system include rapid expression, scalability, and technical simplicity. VectorBuilder specializes in the production of recombinant proteins in S. cerevisiae with a highly optimized workflow for the expression of secretory as well as intracellular proteins. In addition, we can generate recombinant proteins in P. pastoris upon request.

Our typical protein production process starts with construct design which includes fusion partner selection, purification tag selection, and codon optimization. This is followed by gene synthesis and cloning of the yeast recombinant protein expression vector. The vector is electroporated into the appropriate yeast strain and genetically stable clones are screened to identify high producers. A small-batch fermentation is then performed to evaluate protein expression, followed by a large-batch fermentation and subsequent protein purification.

Typical_workflow_of_recombinant_protein_production_in_yeast.jpg

Figure 2. Typical workflow of recombinant protein production in yeast.

Mammalian cells

Producing recombinant proteins in mammalian cells enables proteins to be expressed in their most natural state with all critical post-translational modifications and proper folding. It is therefore the preferred approach for generating therapeutic proteins, vaccines, and antibodies. VectorBuilder specializes in recombinant protein production in HEK293T cells with a highly optimized transient transfection platform for the expression of secretory as well as membrane proteins. In addition, we can generate recombinant proteins in CHO cells upon request.

Our typical protein production process starts with construct design which includes fusion partner selection, purification tag selection, and codon optimization. This is followed by gene synthesis, cloning of the mammalian protein expression vector, and subsequent transfection into HEK293T cells. A small-batch transfection is performed to evaluate protein expression. Based on expression evaluation we then perform a large-batch transfection followed by subsequent protein purification.

Typical_workflow_of_recombinant_protein_production_in_mammalian_cells.jpg

Figure 3. Typical workflow of recombinant protein production in mammalian cells.

Insect cells

Infection of high-density insect cell cultures with baculovirus expressing target genes is another widely used approach for producing recombinant proteins while maintaining majority of eukaryotic post-translational modifications and protein folding. It is often the preferred approach for producing kinases and steroid receptor, which otherwise remain inactive when produced in bacterial cells due to lack of proper phosphorylation or for being misfolded, respectively. VectorBuilder specializes in recombinant protein production in Sf9 insect cells with a highly optimized workflow for the expression of secretory, membrane as well as intracellular proteins.

Our typical protein production process starts with construct design which includes fusion partner selection, purification tag selection, and codon optimization. This is followed by gene synthesis, cloning of the baculovirus transfer vector and subsequent generation of recombinant bacmid. The recombinant bacmid is then transfected into insect cells for generating recombinant baculovirus followed by its amplification. A small-batch transfection is performed to evaluate protein expression. Based on expression evaluation we then perform a large-batch transfection followed by subsequent protein purification.

Typical_workflow_of_recombinant_protein_production_in_insect_cells.jpg

Figure 4. Typical workflow of recombinant protein production in insect cells.

Quality control

All proteins produced by VectorBuilder undergo stringent quality control to ensure that they contain the exact sequence as designed, are free of contaminants, and guaranteed to meet your project specifications. Default QC includes: 1) validation of the protein expression vector by restriction digestion analysis and Sanger sequencing; 2) determination of protein purity by SDS-PAGE and A260/280 measurement. Additionally, we can perform western blot, endotoxin assay, mass spectrometry, and SEC-MALS (combination of size-exclusion chromatography with multi-angle light scattering) analysis upon request.

How to Order

FAQ

Which recombinant protein expression system should I choose?

All recombinant protein expression systems have their own advantages and disadvantages which should be taken into consideration while selecting the optimal system suitable for your project. The table below summarizes the pros and cons of each system.

Recombinant protein expression system Pros Cons
Bacteria
  • Cost-effective.
  • Short production time.
  • Technically simple.
  • Can be scaled up easily.
  • High protein yield.
  • Proteins lack post-translational modifications.
  • Codon usage is different from that of eukaryotes.
  • Difficult to express certain proteins due to solubility issues.
  • Accumulation of inclusion bodies making protein purification difficult.
  • Some proteins are toxic and can inhibit bacterial growth.
Yeast
  • Cost-effective.
  • Short production time.
  • Technically simple.
  • Can be scaled up easily.
  • High protein yield.
  • Incorporates some post-translational modifications such as N-glycosylation, phosphorylation, etc.
  • Suitable for secretory as well as intracellular proteins.
  • Hyper-glycosylation of proteins.
  • Glycosylation pattern is different from that of higher eukaryotes.
  • Presence of large quantities of mannose in N-glycan structures.
Mammalian cells
  • Produces proteins in their most natural state with all necessary post-translation modifications and proper folding.
  • Suitable for secretory and membrane proteins.
  • Most appropriate for producing therapeutic proteins.
  • High production cost.
  • Long production time.
  • Requires complex growth conditions.
  • Scaling up can be difficult.
  • Not suitable for intracellular proteins due to low yield.
Insect cells
  • Performs majority of complex post-translational modifications and protein folding.
  • Suitable for secretory, intracellular, and membrane proteins.
  • Can be used for producing large protein complexes.
  • High levels of protein expression compared to other eukaryotic expression systems.
  • Produced in high-density, suspension cultures making it highly scalable.
  • High production cost.
  • Long production time.
  • Requires complex growth conditions.
  • Technically challenging.
Why is my bacterial vector not expressing my recombinant protein?

Described below are some common reasons for why you might be having issues expressing your recombinant protein in bacteria.

The plasmid is placed in an inappropriate host strain

The plasmid may not be expressed in an inappropriate host strain for induction. Most vectors from VectorBuilder are shipped as E. coli stock in the cloning host strain VB UltraStable™ (this information is also indicated on the vector report). VB UltraStable™ is the preferred cloning host due to its ability to maintain the stability of the plasmid, but it may not be suitable for recombinant protein expression. For example, for pET, the IPTG-induced recombinant protein expression requires the T7 RNA polymerase to be expressed in the host strain, which is not present in VB UltraStable™. As such, bacterial expression vectors typically require transferring the plasmid into an appropriate host strain such as BL21(DE3) for proper induction.

The protein expressed on the vector is “problematic”

Sometimes when the protein being expressed is insoluble, misfolded, improperly cleaved, or toxic to the bacterial host, it may result in poor induction of the recombinant protein. In this case, you will need to optimize your induction system (see below), or express your gene of interest in a more tolerant host strain or from an alternative expression system.

Your induction system is not optimized

Depending on the gene to be expressed, the expression vector and the host strain, you may need to consider optimizing the following factors in your induction system: OD600 (usually between 0.6 and 0.8); concentration of the inducing agent (e.g. IPTG or L-arabinose) cannot be too low or too high; duration of induction cannot be too short or too long; induction temperature needs to be optimized especially when you are dealing with “problematic” proteins (see above), and different temperatures can be tested (e.g. 16°C, 25°C, 30°C, 37°C, etc.). In rare cases, for unknown reasons, different clones of the same expression vector may show different induction behavior, so you may need to pick a number of single colonies to test individually and select the one that has optimal induction performance.

To avoid running into the issues described above and save your valuable time for real research, simply outsource your recombinant protein expression needs to VectorBuilder.