The production of recombinant proteins (or recombinant proteins) is a central part of biotechnology. Recombinant protein expression systems are used for the large-scale production of proteins from recombinant DNA. The process involves the production of proteins using cell cultures, fermentation, or plant cell (chloroplast) expression.
The choice of the recombinant protein expression system depends on factors such as cost, the volume of production required, and regulatory requirements for safety and environmental impact.
The recombinant protein expression system can be further broken down into different types based on the type of cells used and the medium used to grow them. These include prokaryotic cells such as Escherichia coli, yeast, insect cells, plant cells, animal cells such as chicken embryo fibroblasts or mammalian tissue culture cells, hybridoma cell lines; and human cell culture.
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What is Recombinant Protein Expression?
Recombinant proteins are proteins produced by living cells or cultured organisms by the expression of foreign DNA that has been integrated into the host genome. The DNA encodes the amino acid sequence of the desired protein.
The production of recombinant proteins allows the commercial production of therapeutic proteins extracted and purified from bacterial or mammalian (e.g., Chinese Hamster Ovary or CHO) cultures.
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Now that we know what recombinant protein expression is, let us look at some of the most common recombinant protein expression systems used and their pros and cons:
Prokaryotic Protein Expression System
The prokaryotic protein expression system uses bacterial cells such as Escherichia coli as host cells. A DNA sequence encoding the recombinant protein is cloned into an expression vector. This vector is then incubated with the bacterial host cells.
The cells take up the vector and express the recombinant protein within the cell with the aid of an enzyme called protease.
- coli is one of the most commonly used prokaryotic protein expression systems due to:
- its ability to grow in minimal media
- low cost of production
- Easy and economic culturing procedures
- The high-quality recombinant protein produced
- high level of regulation.
There are several downsides to using E. coli, such as:
- Its inability to produce proteins with disulfide bonds/
- Sensitivity to pH
- Lack of folding specificity.
Yeast Proteins Expression System
The yeast protein expression system uses the unicellular, budding yeast species Saccharomyces cerevisiae as host cells. The DNA sequence encoding the recombinant protein is cloned into an expression vector.
This vector is then incubated with the host cells in a medium rich in amino acids. The host cells take up the vector and express the recombinant protein within the cell. Protein expression occurs in the cytoplasm, and the action breaks the cell wall of host proteases. The recombinant protein is then released into the extracellular media.
The main advantages of the yeast protein expression system are:
- Its ability to produce proteins with disulfide bonds.
- It can also produce more proteins than the prokaryotic protein expression system.
The main disadvantages of this system are:
- The high cost of operation.
- Susceptibility to contamination.
- The fact that large volumes of media are used.
Insect Proteins Expression System
The insect protein expression system uses the insect host cells such as Sf9 insect cells or Sf21 insect cells. The DNA sequence encoding the recombinant protein is cloned into an expression vector.
This vector is then incubated in a protein-free insect cell culture medium. The host cells are cultivated in large bioreactors. After some time, the cells take up the vector and express the recombinant protein within the cell.
The advantages of this system include:
- Ability to produce recombinant proteins at a large scale
- High yields of recombinant proteins
- Stable DNA integration
- Easy purification of recombinant protein cells
- Low production costs.
The downside to this system is:
- Susceptibility to contamination and the need to use feeder cells to keep the insect cells alive.
Plant Proteins Expression System
The plant protein expression system uses the unicellular chloroplast. The DNA sequence encoding the recombinant protein is cloned into an expression vector. This vector is then incubated with the chloroplasts isolated from the plant species such as tobacco, alfalfa, or corn. The chloroplasts take up the vector and express the recombinant protein within the chloroplast.
The main advantages of the plant protein expression system are:
- Ability to produce proteins with disulfide bonds that cannot be produced in bacteria or yeast expression systems
- Low cost of operation and safety.
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The main drawbacks of this system are:
- The low yields.
- Chloroplasts from only a few plant species are used.
- The use of large volumes of media.
- The fact that only a few plant species are used.
- Compared to bacterial and yeast expression systems, it takes longer for plant seeds to grow and mature, which may cause issues when producing large batches of protein.
- The low solubility of many amino acids compared to bacterial systems-This causes issues integrating ingredients into the cell membrane and the need for post-translational modifications to improve solubility.
Choosing the right expression system can be challenging as they all have advantages and disadvantages. Bacterial expression systems are ideal for co-expression and secretion. They are therefore helpful for producing recombinant proteins such as antibodies and hormones naturally secreted by cells.
As with any other process, the success of recombinant protein expression is heavily dependent on the experience of the person responsible for the procedure.
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