What is the Advantage and Disadvantage of gmp recombinant proteins

Recombinant protein expression enables the production of biological therapies, vaccines, or ancillary reagents, among others. A critical decision in any project is the choice of the right host system, as each comes with advantages and disadvantages that can impact yield, quality, and cost. In this article, we explore the most commonly used host systems in recombinant protein production.

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Bacteria: A Versatile and Efficient Option

Regarding bacterial hosts, we will analyze the two most commonly used: E. coli and Bacillus subtilis. According to various studies, bacteria are the most frequently chosen systems due to their rapid growth and scalability.

Escherichia coli is one of the most widely used host systems for recombinant protein expression due to its fast growth, ease of genetic manipulation, and low cost. This organism can achieve high production yields in a short time, making it ideal for projects requiring speed and scalability. However, one major challenge with E. coli is its inability to perform complex post-translational modifications, such as glycosylation, which are essential for the activity and stability of certain biotherapeutic proteins. Additionally, proteins expressed in E. coli are often found in the form of inclusion bodies, requiring additional steps for solubilization and refolding. Endotoxins present in the cell membrane can also limit its application in human biopharmaceuticals.

Bacillus subtilis is another bacterial host popular for protein production, particularly due to its ability to secrete proteins directly into the culture medium, simplifying recovery and purification. Furthermore, this microorganism does not produce endotoxins, making it an attractive option for therapeutic applications. Nevertheless, Bacillus has some limitations, such as the production of proteases that can degrade recombinant proteins, and its genetic manipulation is more complex compared to E. coli.

Yeasts: Balancing Productivity and Post-Translational Modifications

For yeast-based systems, two species stand out: Pichia pastoris and Saccharomyces cerevisiae. Yeasts are widely recognized for their balance between cost-effectiveness and their ability to perform post-translational modifications.  Pichia pastoris is a widely used host system for recombinant protein expression due to its ability to grow to high cell densities in bioreactors, increasing productivity. It can perform certain post-translational modifications, such as glycosylation, that are not possible in bacteria. This makes it an ideal choice for proteins requiring proper folding and biological functionality. However, the glycosylation patterns in Pichia differ from those in mammalian systems, which can limit its use for some biopharmaceutical products. Additionally, the use of methanol as an inducer for the most common promoters involves higher operational costs and safety considerations.

Known as baker’s or brewer’s yeast, Saccharomyces cerevisiae is one of the most studied and utilized systems in biotechnology. It offers the ability to perform post-translational modifications and has been approved for the production of numerous biologics, including vaccines. However, S. cerevisiae tends to hyper-glycosylate proteins, which can be problematic for certain therapeutic products. Additionally, its secretion of proteins into the medium is limited, complicating purification.

Filamentous Fungi: High-Yield Production

When discussing filamentous fungi, two key systems are Trichoderma reesei and C1 (Myceliophthora thermophila). These systems are especially noted for their capacity to produce industrial enzymes. Learn more about the C1 platform’s innovative capabilities on Dyadic’s official website. Trichoderma reesei is renowned for its high secretion capacity, making it ideal for industrial applications requiring mass production. Its ability to produce industrial enzymes is well-recognized.

Like Pichia pastoris, Trichoderma exhibits glycosylation patterns that differ from mammalian systems, limiting its use in pharmaceutical products. Moreover, its genetic manipulation is more challenging and requires advanced techniques.

The C1 system is a promising filamentous fungus due to its ability to produce exceptionally high amounts of protein. Its simple cultivation requirements reduce operational costs. Despite its advantages, C1 is relatively new compared to other host systems, which may lead to hesitancy in adoption due to a lack of validation in certain applications.

Choosing the right host system for recombinant protein expression depends on several factors, as a summary:

At 53Biologics, we specialize in customized strain development to optimize protein expression across various host systems. To learn more about our services and how we can help you achieve your goals, visit our Strain Development page.

About 53Biologics:

53Biologics is a Spanish CDMO specialized in decoding biologics production, from DNA to proteins. The company provide services from preclinical development to GMP manufacturing, supporting their clients in getting their biological products to market as quickly as possible.

Written by Sophie Williams, PhD researcher at the University of Bristol

The insertion of protein tags into recombinant proteins has become a useful and powerful tool in protein research. These tags can aid in the purification of the target protein from cell lysate, monitor the localization of the protein intracellularly, and aid in the detection of a previously uncharacterized protein. To date, there is a wide range of affinity tags to choose from, but can adding a non-native peptide or fusion protein tag have adverse effects?

Various protein tags are used routinely in research, including larger fusion-protein tags such as green fluorescent protein (GFP), maltose binding protein (MBP), and glutathione S-transferase (GST). Alternatively, smaller, more discrete peptides may be inserted into the recombinant target protein including a poly-histidine tag (His-tag) or a strep-tag. These tags are commonly added to either the N- or C-terminus of the target protein depending on the properties of the protein (e.g., the presence of subcellular localization sequences, downstream cleavage sites, or post-translational modification sites).

Applications of protein tags

When considering which protein tag to use in your research system, there are a few factors to consider:

• Impact of the tag on the target protein’s structure and function

• Requirement of a linker to separate the tag from the protein

• Tag location

• Inclusion of a protease cleavage site for tag removal

• Final application of the protein

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• Required production level of recombinant protein

Along with this, some tags are more suited for specific purposes by incorporating desirable properties into your target protein.

Stability improvements

The production of tricky proteins, such as those requiring post-translational modifications, can be particularly troublesome in common expression hosts such as E. coli. As E. coli lacks the sophisticated machinery required for post-translational modifications found in many eukaryotic proteins, the expression of eukaryotic proteins in E. coli often results in protein misfolding and the production of inclusion bodies. As such, although up to 75% of human proteins can be expressed in E. coli, only 25% can be produced in the functional, soluble form1.

The addition of some protein tags may aid in the stability issues of troublesome proteins. For example, tags such as MBP, GST, and small ubiquitin related modifier (SUMO) are routinely fused with difficult proteins with poor solubility. These larger protein tags fold rapidly into stable proteins with good solubility after translation. The rapid folding of the protein tag can drive the folding of the target protein, aiding in the solubility and stability of the target protein while preventing its intracellular aggregation. This can be particularly important if high yields of recombinant protein are desired, such as during protein purification. For example, the human interferon IFN-γ was recently successfully expressed and purified in a functional form following SUMO-fusion, whereas previously it has aggregated into inclusion bodies in the native form2.

Protein-tag fusions can also aid in membrane protein production. Membrane proteins are traditionally complicated to express and purify, often becoming toxic to the expression host when overexpressed and requiring extraction from the cell membrane with detergents. They are also often unstable; however, it has been demonstrated that the fusion of protein tags may aid in membrane protein stability. For example, MBP has been fused to eukaryotic G-protein-coupled receptors to aid in expression and SUMO tags have been shown to aid in the folding and stability of both prokaryotic and eukaryotic membrane proteins3,4.

Figure 1. Examples of fusion protein tags to membrane protein purification. Figure taken from Liu and Li, Crystals (, open source)3.

Fluorescent signal

GFP and other related fluorescent protein tags provide a direct method to carry out subcellular localization studies relatively quickly and simply when applied with fluorescence imaging techniques. These fluorescent probes may be fused to the target protein to monitor its localization, abundance, and movements intracellularly with high spatial and temporal resolution.

Detection

GFP along with non-fluorescent tags such as FLAG® (DYKDDDDK), c-myc, or hemagglutinin antigen (HA) tags are widely used for protein detection during IP and western blotting studies. This can be particularly useful when working with an uncharacterized protein or a protein to which a good antibody has yet to be developed. Antibodies and Nanobodies to these tags are readily available and work excellently in western blotting, IP, and affinity purification.

Affinity

Fusion tags with high affinities to interaction partners such as small molecules (e.g., metal ions) or proteins (e.g., streptavidin, antibodies) may provide a useful method to purify the recombinant protein relatively simply and quickly. Affinity chromatography exploits this characteristic of numerous protein tags including the His-tag, Strep-tag, and GST. For example, the His-tag binds with high affinity to transition metal ions such as Ni2+ or Co2+, which can be immobilized on a solid support such as beads or resin. Once the His-tagged protein is bound to the metal-resin, contaminating proteins can be washed away leaving the desired protein that may be eluted in a high imidazole concentration buffer.

Drawbacks of inserting protein tags

Although the use of protein tags has revolutionized protein science, certain drawbacks need to be considered before tags are added:

1. Unknown impacts on the target protein

The insertion of protein tags (especially larger tags) may have unknown impacts on the structural and functional characteristics of the target protein. The insertion of a tag can lead to either partial or complete loss of function. For example, although small in tag-size, the insertion of a His-tag has resulted in significant reductions in activity in multiple enzymes in recent studies2,5. Therefore, to determine if the tag fusion is having an impact on the activity of the target protein, complementation studies to monitor the wild-type versus the recombinant protein’s activities would be required.  

2. Protein instability upon tag removal

The removal of solubilizing fusion tags for downstream applications such as crystallization of the target protein may bring about the return of the target protein’s instability. As such, co-expression with a chaperone or antibody fragment binding may be a more appropriate approach if tag removal is required. Chaperones assist newly synthesized proteins in their correct folding while burying the target protein’s hydrophobic regions to prevent protein aggregation. As such, chaperones such as protein disulfide isomerase (PDI), GroES/L, or trigger factor (TF) are commonly co-expressed with troublesome proteins.

3. Extra steps of tag removal

Efficient removal of protein tags from the recombinant protein can often be difficult and time-consuming. Typically, this involves the use of a protease and the insertion of a protease cleavage site between the tag and protein, but the cleavage sequence may also influence the structure or function of the target protein.

4. Incorporation of non-native sequences

Some biotherapeutics require all protein tags to be removed (including linkers and protease cleavage sites) to leave only the native sequence. The presence of non-native sequences may interfere with the therapeutics function or may result in an immune response being raised when the drug is administered. As such, Proteintech’s Humankine® RUO and cGMP recombinant cytokines and growth factors are always tag-free.

Summary

Overall, each protein tag comes with its own benefits and hindrances. However, when the application and the target system are carefully considered, protein tags can provide an invaluable tool, saving researchers time when tackling tricky proteins.

Nanobodies against tags

Nano-Traps: affinity beads for immunoprecipitation

ChromoTek Nano-Traps are the benchmark in immunoprecipitation (IP) and allow fast and reliable one-step pulldowns of even low expressed proteins. They consist of Nanobodies coupled to beads and are ready-to-use. Low background, no extra bands & high specificity will improve your pulldown assay significantly.

Watch why GFP-Trap® gives the best results for immunoprecipitation:

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Antibodies against tags

Highly cited antibodies against all popular tags. Browse here.

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