Custom Protein Service

Service Features

• ✔ Comprehensive. From gene synthesis to protein purification, and beyond (desalting, aliquoting, lyophilization, conjugation)
• ✔ Fast Turnaround. 3 weeks for bacterial expression; 8 weeks for mammalian cell expression.
• ✔ Easily Scalable. Once the protein has been purified for the first time, we can scale up production to your specifications.
• ✔ High Purity. Typically >95% purity as determined with SDS-PAGE
• ✔ Affordable.

Gene Expression Systems

RayBiotech offers bacterial or mammalian gene expression systems to serve a variety of research requirements.

Bacterial gene expression

• Cell type: E. coli
• Expression level: Low-Medium-High
• Cell doubling time: Rapid (30 min)
• Cost: Lowest
• Timeline: 3 weeks
• Post translational modifications (PTMs): None
• Summary: Bacterial gene expression is a fast and cost-effective approach for producing recombinant proteins.

Baculovirus insect cell expression

• Cell type: Sf9, HighFive
• Expression level: Low-Medium-High
• Cell doubling time: Slow (18-24 hr)
• Cost: Low-High
• Timeline: 6-8 weeks
• Post translational modifications (PTMs): Yes
• Summary: Baculovirus insect cell gene expression is an ease of scale-up from the laboratory to a large-scale production system. The expressed proteins have the abundant post-translational modification.

Mammalian gene expression

• Cell type: Human HEK293 suspension cells (TruExp^™ Mammalian Cell Gene Expression System)
• Expression level: Low-Medium-High
• Cell doubling time: Slow (24 hr)
• Cost: Low
• Timeline: 8 weeks
• Post translational modifications (PTMs): Yes
• Summary: Mammalian gene expression is the best option for producing native conformations and post translational modifications of mammalian proteins.

RayBiotech TruExp^™ Mammalian Cell Gene Expression System

TruExp^™ is a state-of-the-art mammalian cell-based expression system developed by RayBiotech for producing recombinant proteins with native conformations and PTMs. TruExp^™ is an excellent option for protein that have a high molecular weight or are difficult to express.

• ✔ Complex, abundant PTMs. See Figure 1
• ✔ High Expression. Typical yield ranges from 2 - 355 mg purified protein per liter of culture (Figure 1, Table 1).
• ✔ Secretion. Expressed protein is secreted into the culture medium, facilitating high purity levels (in contrast to recovering protein from highly contaminated lysate fractions) (Figures 1 & 2).
• ✔ No Biohazard. Serum-free medium and animal source-free materials eliminate biohazard risk.

Table 1.Typical recombinant human protein yields of Raybiotech TruExp Mammalian Cell Gene Expression System

Protein Name		Yield (mg/L culture)
Serpin B4, Serpin Peptidase Inhibitor	SCCA2	335.7
Calcitonin	CT	160.8
14-3-3 Protein Epsilon	YWHAE	103.1
N-terminal of the prohormone BNP	NT-proBNP	99.5
Lipocalin-2	NGAL	69.1
a-Fetoprotein	AFP	63.2
Brain natriuretic peptide 32	BNP-32	51.2
Ubiquilin-1	UBQN	41.3
Carcinoembryonic Antigen	CEA	39.8
Receptor Tyrosine-Protein Kinase erb-3	erbB-3	37.5
Epidermal Growth Factor Receptor	EGFR	23.5
Pepsinogen II, Pepsinogen C	PGC	18.6
Lymphocyte Activation Gene 3 Protein	LAG-3	16.9
Receptor Tyrosine-Protein Kinase erbB-2	Erb/Her2	16.7
Hepatocyte Growth Factor Receptor	HGF R	14.6
Annexin A2	ANXA2	11.8
Golgi Membrane Protein 1	GP73	5.5

Table 2. The advantages and disadvantages of different gene expression systems

Protein Expression Service Overview

Phase 1: Gene synthesis

• Lead time: Depends on gene size. Usually 2 - 4 weeks.
• Description: The target gene is synthesized, which is codon optimized for the expression system that will be used.

Phase 2: Gene cloning

• Lead time: 1 week
• Description: The target gene is cloned into RayBiotech OVER-expression vectors with an N-terminal signal peptide for protein secretion and an N-terminal or C-terminal 6x Histidine (His) tag for protein affinity purification. The recombinant construct is confirmed by DNA sequencing.

Phase 3: Small-scale gene expression

• Lead time: 1 - 2 weeks
• Description: The plasmid cDNA is extracted from the cells and transfected into 5 ml of E. coli or 2 ml of human HEK293 suspension cells. The target protein in the cell lysate and supernatant is analyzed via SDS-PAGE and/or western blotting using an anti-His tag antibody.
• Deliverable: The customer is sent the SDS-PAGE and/or western blotting results electronically.

This phase evaluates gene expression, protein expression levels, protein secretion, and protein solubility. If the protein does not meet the customer's expectation, the service can be stopped after this phase.

Phase 4: Large-scale gene expression and purification

• Lead time: 2 - 4 weeks
• Description: The plasmid cDNA is extracted from the cells and transfected into 2 liters of E. coli or 500 ml of HEK293 suspension cells (3 million cells per ml). The cells are cultured. The target protein is purified via its His tag and immobilized metal ion affinity chromatography (IMAC). The concentration and purity of the purified protein are determined using a bicinchoninic acid (BCA) assay and SDS-PAGE with Coomassie blue staining, respectively. The protein is desalted into 0.2 µm filtered 1x phosphate-buffered saline (PBS), pH 7.4. The protein can then be aliquoted, lyophilized, and labeled upon request (additional lead time may apply).
• Deliverable: The customer is sent all the protein purified in this phase.

Phase 5: Bulk production (optional)

• Lead time: Depends on the amount of protein required.
• Description: Large-scale gene expression and purification based on the customer's desired amount of purified protein.
• Deliverable: The customer is sent all the protein purified in this phase.

Service FAQs

• How much does the service cost?

  The price is based on a customer's specific requirements and will vary from customer-to-customer. For a quote tailored to your needs, complete the quote request form.

• Does RayBiotech guarantee that the target protein is correct and will be expressed?

  We guarantee that your target gene-of-interest is correctly synthesized using DNA sequencing.

  We cannot guarantee that your protein will be expressed since expression level cannot be predicted. This is why a small-scale gene expression test to determine protein expression is performed in Phase 3. However, it is known that membrane proteins are often poorly expressed and purified. Therefore, we recommend that only soluble domains are expressed.

  You only need to pay for the work that is performed. That is, if the results in Phase 3 shows that the expression failed and you choose not to continue to Phase 4, you do not need to pay for Phase 4.

• Why does RayBiotech use a signal peptide for secretion?

  The signal peptide causes the protein target to be secreted out of the cell and into the serum-free culture media where the protein content is minimal. This will result in a purer purified protein since there are fewer proteins in the culture supernatant (compared to the cell lysate) to nonspecifically bind to the affinity purification column. For example, protein purity > 95% is routinely achieved with secreted proteins, whereas protein purity > 50% is often obtained with non-secreted proteins.

  Sometimes the protein target will not be secreted even with the signal peptide. It is likely that, in the protein tertiary structure, the signal peptide is occluded.

  The signal peptide is 25 amino acids long. It is often cleaved by proteases in cell culture, but we cannot guarantee that the signal peptide will be cleaved. Upon request, we can exclude the signal peptide in the expressed protein.

• Can I use my own expression vector?

  Yes. If you have already expressed your target protein successfully using your expression vector, please provide the expression conditions that you would like us to use. We can then go to Phase 4 directly. If you have not confirmed that the gene is expressed in your expression vector, then we will perform a small-scale expression test in Phase 3.

• I already have my target gene in a plasmid. Can the gene be moved into the RayBiotech's expression vector?

  It depends. You will need to provide us with the gene sequence and, at a minimum, 100 nucleotides flanking the gene at the 5' and 3' ends. This information is necessary because we will use restriction enzymes to cut the gene out of your plasmid and ligate it into our ours (i.e., cloning). If we can clone your gene into our vector, then Phase 1 is not necessary. If the gene and flanking sequences are incompatible with any restriction digest approach, then we cannot use your gene and will have to start at Phase 1.

  Also, it is important to consider whether your gene is codon optimized for the expression system.

• I don't want a His tag. Can a different tag for purification be added?

  Yes, we can. Please indicate this information in the quote request form.

• I don't want any tag in my final purified protein.

  We can add a cleavage site between the tag and target protein.

  For example, we can add a tobacco etch virus (TEV) site between the His tag and protein-of-interest. After the His-tagged protein is purified, a His-tagged protease could be added, cleaving the His tag from the target protein. The tagless protein could then be purified from the His tag and His-tagged TEV protease using IMAC.

• I previously had a custom protein expressed and purified by RayBiotech. I now need more. What should I do?

  If the protein was expressed and purified previously, you do not need to pay for Phases 1 - 4 again. You would simply complete the quote request form for the desired amount of purified protein (Phase 5). Please make sure to include the previous sales order or invoice number in the Project Description section.