Recombinant Protein Expression Systems

In any investigation in the field of proteomics, the production of the protein of interest is usually necessary to carry out structural, functional, locational or interaction studies between proteins.

The expression systems of recombinant proteins possible to obtain large amounts of protein with homologous to native characteristics by genetic engineering and cell culture, in a process that includes:

  • Construction of the vector with the sequence that encodes the protein of interest
  • Transfection of cells with said vector
  • Translation of those genes into proteins using the cellular machinery of the host cell
  • Cell lysate, extraction and purification of the protein of interest

In this post we tell you about the advantages and disadvantages of each of the recombinant protein expression systems, which will allow you to select the most appropriate system based on the type of protein, performance and application, among others.

1.- BACTERIA

The bacteria that are most frequently used as recombinant protein expression systems are those of the Escherichia coli species , although the use of other strains such as Bacillus subtillis is also common .

  • Advantage
    • Allows the production of large amounts of protein in a short period of time
    • Transformation protocols are simple
    • Requires simple growing conditions
    • Fast growth
    • Low cost
    • Scalable process
    • It allows modifying various parameters to optimize expression levels
  • Disadvantages
    • Does not offer post-translational modifications
    • Accumulation of proteins in inclusion bodies
    • Protease contamination of the host cell
    • Accumulation of endotoxins
    • Poor performance for some eukaryotic proteins
    • Difficulty expressing high molecular weight proteins
2.- YEASTS

Among the yeasts that are most frequently used as recombinant protein expression systems, Pichia pastoris and Saccharomyces cerevisiae stand out .

  • Advantage
    • Well defined and inexpensive eukaryotic expression system.
    • Allows the expression of both secreted and intracellular proteins
    • High performance
    • Low expression time
    • Requires simple culture media
    • Offers post-translational modifications
    • Scalable process
    • Endotoxin free
  • Disadvantages
    • Protein hyperglycosylation
    • Glycosylation different from mammalian cells
3.- INSECT CELLS

It is a widely used technique to express mammalian proteins that require post-translational modifications, and can be used in both adherent and suspension cultures.

  • Advantage
    • Simple purification process
    • Good expression levels, especially for intracellular proteins
    • Relatively fast growth
    • Endotoxin free
  •  Disadvantages
    • The cloning process of the gene of interest in the baculovirus vector is time consuming
    • The scaling process requires large volumes of viruses
    • Expensive culture medium
    • Glycosylation is different from that achieved in mammalian systems (although more similar than that produced by yeasts)
4.- MAMMALIAN CELLS

Among the most common mammalian cell lines such as recombinant protein expression systems, CHO (Chinese HAmster Ovarias) and HEK 293 (Human Embryonic Kidney) cells stand out.

It is the system of choice for the expression of vaccines and therapeutic proteins, and can be obtained from both transitory and stable cell cultures.

  • Advantage
    • Offers all post-translational modifications
    • Correct folding of the protein
    • Endotoxin free
  • Disadvantages
    • High cost because the cells grow very slowly, the culture medium is expensive and requires a constant supply of CO2 and expensive transfection reagents
    • Relatively low performance
    • Longer production time
5.- CELL-FREE SYSTEMS

The most common systems include wheat germ or rabbit reticulocyte expression systems.

  • Advantage
    • Simple process
    • Scalable
    • Proteins can be purified in a very short period of time
    • Allows labeling of proteins for structural studies
  • Disadvantages
    • Degradation of proteins by exogenous proteases
    • Cell extracts are expensive

In conclusion, the recombinant protein expression systems will be chosen based on the objective pursued, the most common prokaryotic systems for the production of antigens, structural and activity studies (which do not require post-translational modifications), and the eukaryotes for the production of vaccines, therapeutic proteins and proteins that require post-translational modifications to exercise their biological activity.