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Recombinant Protein Expression in Mammalian Cells & E.coli Recombinant Protein Expression in Mammalian Cells & E.coli

Recombinant Protein Expression

  • Basic Research
  • Therapeutic Development
  • Starting from 2 weeks

Overview

Expression systems utilizing mammalian cells for recombinant protein manufacture introduces proper protein folding, post-translational modifications, and protein assembly, which are important for producing proteins of complete biological activity, and thus is in high demand in the therapeutic development and application fields, especially for biotechnology and pharmaceutical companies. Besides industry use, academic research groups can also rely on recombinant mammalian proteins for functional studies and high-resolution structure determination.


Biointron, with high flexibility, good cost-effectiveness, and big production capabilities, offers high-quality protein expression service via mammalian cell system using CHOKl and HEK293 cells.

Recombinant Protein Expression in Mammalian Cells & E.coli Overview

How Expression in
Mammalian Cells Work

Expression via mammalian cell system technology generally begins with the synthesis of your sequence of choice. Following the construction of plasmid vectors, we conduct transfection of the recombinant vector into CHOK1 (Chinese hamster ovary) cells or HEK293 (human embryonic kidney) cells for protein expression and purification, with stringent quality control measures SDS-PAGE analysis to confirm its presence and purity.

How Expression in
E. coli Works

Expression via E. coli is a multi-step process that begins with the design and construction of a plasmid carrying the gene sequence for the desired protein. Following this, the plasmid is transformed into the target host cell, which are then cultured under controlled conditions to promote growth and induce protein expression. After reaching an optimal expression level, the cells are lysed to release the protein, which is then purified using various techniques including chromatography.

Applications

Expression via mammalian cells is the dominant recombinant protein production system for clinical uses, with hundreds of biopharmaceutical product candidates in clinical development.

Basic Research

  • Signals for synthesis, processing and secretion of eukaryotic proteins are correctly recognized by mammalian cells
  • Allows for proper protein folding, glycosylation, and post-translational modifications, which are often required for full biological activity with in vitro and in vivo systems
  • Protein structure, function, and characterization research

Therapeutic Development

  • Production of monoclonal antibodies (mAbs), antibody screening, and antibody libraries
  • For use with cell lines, vectors and transfection methodology
  • Production of important clinically active viral surface antigens
  • Biotechnology research fields including immunology and oncology applications

Highlights

Basic Research

  • Signals for synthesis, processing and secretion of eukaryotic proteins are correctly recognized by mammalian cells
  • Allows for proper protein folding, glycosylation, and post-translational modifications, which are often required for full biological activity with in vitro and in vivo systems
  • Protein structure, function, and characterization research

Therapeutic Development

  • Production of monoclonal antibodies (mAbs), antibody screening, and antibody libraries
  • For use with cell lines, vectors and transfection methodology
  • Production of important clinically active viral surface antigens
  • Biotechnology research fields including immunology and oncology applications

Fast Turnaround Time

  • Only 2-3 weeks for expression via mammalian cell system using CHOK1 or HEK293 cells
  • Expression via mammalian cells is the dominant recombinant protein production system for clinical uses
  • Hundreds of biopharmaceutical product candidates are in clinical development
Speak to A Specialist

Working Flowchart

Gene Synthesis

Plasmid construction and preparation

Transient expression

Affinity purification

Stringent quality control

Fast delivery

Expression in Mammalian Cells
Service Details

Service Step Service Description Timeline Deliverables
Gene synthesis
  • Codon optimization and gene synthesis
  • Subcloning into an expression vector
  • Plasmid amplification and preparation
 2 to 3 weeks
  • Expression plasmid contain GOI
  • Purified Recombinant protein (SDS-PAGE and SEC-HPLC detection, endotoxin level <1EU/mg)
  • COA report
100ml Pilot study
  • Transfection of mammalian cells
  • 100ml pilot protein expression
  • QC analysis
Scale up production(1L-10L)
  • Large scale protein expression
  • Affinity purification
  • QC analysis
 3 to 4 weeks
  • Purified Recombinant protein (SDS-PAGE and SEC-HPLC detection, endotoxin level <1EU/mg)

Case Study

  • Case 1: PD-L1 (Fc tag), Extracellular domain (19Phe-239Thr) expressed in CHO
    SDS-PAGE

    R: Reducing condition;

    N-R: Non-reducing condition;

    M: Marker

    Detector A Channel 2 280nm
  • Case 2: CD137 (His tag), Extracellular domain (Glu19-Sr184) expressed in HEK293
    recombinant-case2-1

    R: reducing condition;
    M: Marker

    recombinant-case2-2

    Human CD137L/mFC; Human CD137/ His;
    Recombinant Human CD137/His is captured on Protein A chip, can bind to Human CD137L/mFc.

    recombinant-case2-3

    Log Human CD137, His tag EC50=4ng/ml
    CD137 is immobilized at 2µg/mL (100 µL/well), it can bind to Utomilumab. The concentration for 50% of maximal effect(EC50) of is 4 ng/ml.

    recombinant-case2-4

    Log Human CD137, His tag EC50=20ng/ml
    CD137 is immobilized at 2µg/mL (100 µL/well), it can bind to Human CD137L. The concentration for 50% of maximal effect(EC50) of is 20 ng/ml.

“We tailor our services according to the client’s needs – we offer the use of either CHOK1 or HEK293 cells for recombinant protein expression. Every successful expression is a step closer to redefining the possibilities in biotechnology and, ultimately, in improving lives.”
Yiyang Ge
Yiyang Ge
Biointron Scientist

FAQs

  • Why use mammalian cell-based protein expression?

    Using a mammalian host system is ideal for producing recombinant proteins in the most physiologically native structure and environment, as it allows for post-translational modifications, such as glycosylation and phosphorylation, or proper folding, if needed for therapeutic use in humans.

  • What is the difference between CHO-K1 or HEK293 cells?

    Biointron offers both CHO-K1 and HEK293 cells for expression via mammalian cell systems, depending on your needs. Typically, we recommend using HEK293 cells for fusion proteins, for better purity. However, for antibodies, we generally recommend CHO-K1 cells for a higher yield.

  • Why are CHO cells frequently used for mammalian protein expression?

    CHO cells are suspension-adapted and can grow well in basic serum-free CD media, thus permitting volume scalability and safety from potential contamination through serums. CHO cells also provide a high yield and cell density, as well as being amenable to genetic manipulation.3,4

Resources

References

  • Malm, M., Kuo, C., Barzadd, M. M., Mebrahtu, A., Wistbacka, N., Razavi, R., Volk, A., Lundqvist, M., Kotol, D., Tegel, H., Hober, S., Edfors, F., Gräslund, T., Chotteau, V., Field, R., Varley, P. G., Roth, R. G., Lewis, N. E., Hatton, D., . . . Rockberg, J. (2022). Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins. Metabolic Engineering, 72, 171-187. https://doi.org/10.1016/j.ymben.2022.03.009
  • Haldankar, R., Li, D., Saremi, Z. et al. Serum-free suspensin large-scale transient transfection of CHO cells in WAVE bioreactors. Mol Biotechnol 34, 191–199 (2006). https://doi.org/10.1385/MB:34:2:191
  • Khan, K. H. (2013). Gene Expression in Mammalian Cells and its Applications. Advanced Pharmaceutical Bulletin, 3(2), 257-263. https://doi.org/10.5681/apb.2013.042
  • Gray, D. (1997). Overview of Protein Expression by Mammalian Cells. Current Protocols in Protein Science, 10(1), 591-5918. https://doi.org/10.1002/0471140864.ps0509s10

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