Custom Protein Service

Expression and purification services of recombinant proteins

Since 2006, RayBiotech has provided Custom Protein Services to produce recombinant proteins based on a customer's specifications. Our comprehensive service program covers every step of production, from gene cloning through protein purification. We can also perform downstream processing like protein desalting, lyophilization, vialing, and labeling.

Protein molecule

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.
Custom Protein
Figure 1. Raybiotech TruExp Mammalian Cell Gene Expression System demonstrates gene over-expression and secretion. Six human genes (H1-6) and 3 mouse genes (M1-3) were expressed by the TruExp expression system. Cell culture supernatants (10 µL) were applied to 12% SDS-PAGE (A) and Western blot probed with anti-his tag monoclonal antibody (B). Except for genes H6 and M2, all overexpressed genes display additional secreted protein bands (A, red arrows). All genes were expressed, with some displaying abundant post-translational modifications, evident by increased molecular weights compared to the predicted sizes (B, yellow arrows). M = protein standard markers (kDa).
Custom Protein
Figure 2. Representative purified human proteins from Raybiotech TruExp Mammalian Cell Gene Expression System. Affinity-purified recombinant proteins from culture supernatants of transfected mammalian cells exhibit high purity and intact protein bands, even at MW larger than 100 kDa. M = protein standard markers (kDa).

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

Characteristic Bacteria Yeast Baculovirus Insect Cells Mammalian Cells
Expression Level Low-Medium-High Low-High Low-High Low-Medium-High
Cell Growth Rapid (30 min) Rapid (90 min) Slow (18-24 hr) Slow (24 hr)
Medium Complexity Simple Simple Complex Complex
Cost Low Low Low-High High
Timeline 3 weeks 3-4 weeks 6-8 weeks 4-5 weeks
Post-Translational Modifications (PTMs)
Protein Folding Not reliable Usually reliable Very reliable Very reliable
Glycosylation None Yes. Not identical to mammalian system. High mannose. Yes. Not identical to mammalian system. Simple, no sialic acid. Yes. Complex/Authentic
Phosphorylation None Yes Yes Yes
Actylation No Yes Yes Yes
Acylation No Yes Yes Yes
Gamma-Carboxylation No No No Yes

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?
  • Does RayBiotech guarantee that the target protein is correct and will be expressed?
  • Why does RayBiotech use a signal peptide for secretion?
  • Can I use my own expression vector?
  • I already have my target gene in a plasmid. Can the gene be moved into the RayBiotech's expression vector?
  • I don't want a His tag. Can a different tag for purification be added?
  • I don't want any tag in my final purified protein.
  • I previously had a custom protein expressed and purified by RayBiotech. I now need more. What should I do?