The study of proteins is important for various medical and industrial purposes. Protein expression is a technique used for manufacturing important proteins. In this article, we will give you a brief overview of how protein expression is done in lab settings – keep reading!
Protein expression: An intro
Cells are responsible for the regulation and production of proteins. Complete information about a specific protein is stored in a cell in the form of DNA. Transcription is responsible for reading and understanding the information stored in DNA regarding a protein.
The transcription results in the production of messenger ribonucleic acid or mRNA. This mRNA then undergoes the process of translation, which produces a specific protein.
Prokaryotic cells are capable of simultaneous transcription and translation, called “coupled transcription and translation.”
On the other hand, Eukaryotic cells can manage the processes of transcription and translation differently. Both transcription and translation are carried out in different spaces. The nucleus is responsible for the transcription process in eukaryotic cells, and protein synthesis is carried out in the cytoplasm.
The transcription process
In terms of sequence, both prokaryotic and eukaryotic cells handle the transcription process in three distinct steps:
- Initiation
- Elongation
- Termination
The transcription process starts with the unwinding of DNA. After unbinding is complete, the DNA can bind with RNA polymerase.
There is a major difference in the transcription process between prokaryotes and eukaryotes. Experimental evidence shows that eukaryotic cells use activators and repressors to handle transcription. Prokaryotic cells, on the other hand, don’t need such modifiers.
The translation process
The translation process is done in several steps and requires molecules called macromolecules. Some of the macromolecules needed in the translation are:
- Ribosomes
- Transfer RNAs
- Messenger RNAs
- Amino acids
- ATP
- GTP
In principle, the basics of the translation process are the same in prokaryotic and eukaryotic cells, but there are some noticeable differences.
The initiation process
A ribosome is bound to initiator tRNA that works with the initiator codon in the initiation process. The reaction between large and small ribosomal units gives rise to the initiation process. The specific structure of mRNA allows the recognition of initiation codon and initiation complex.
Elongation is a process that is included in the initiation process. Some amino acids also add up to the growing peptide and play their part in sequencing transcription. After that, the ribosome gets released from mRNA and starts the process of translation.
The post-translational modification
In essence, post-translational modifications are alterations that are carried out inside the cell. Modification of polypeptides starts after the process of translation is complete. This modification process makes it easier for the cell to identify polypeptides and regulate them properly.
There are several types of post-translational modifications that are arranged within the cell. For example, a post-translational modification is a process in which a polypeptide gets folded into a globular protein to attain its lowest energy state.
Some important types of PTMs are:
- Phosphorylation
- Ubiquitination
- Nitrosylation
- Acetylation
- Lipidation
Protein expression through recombinant methods
The study of proteins for medicinal or industrial uses revolves around investigating a protein’s structure or its interactions with other proteins. Scientists rely on protein expression methods to extract specific information about a protein.
It’s comparatively difficult to study complex proteins by producing them through chemical synthesis. Therefore, scientists rely on living cells of organisms to study how specific proteins are manufactured and handled within a cell.
Several mechanisms can be employed to study protein expression in eukaryotic and prokaryotic cells. However, the selection of the mechanism and the type of cell chosen to study a protein depends on the protein being examined.
Here, we give a brief introduction to protein expression systems that researchers around the globe employ.
1. Through mammals
The best way to study mammalian proteins is to study them under mammalian expression systems. Due to their physical superiority, the mammalian physiological environment is better for studying proteins instead of relying on chemical synthesis.
The biggest benefit of using mammals for protein expression is that it allows producing antibodies easily. Two methods can be used for protein production through mammalian expression, namely:
- Transient process
- Stable cell lines
The expression construct is introduced into the host genome to carry out the protein expression process in both these processes. The choice of transient or stable cell lines for protein expression depends on the desired outcome.
For example, if a researcher wants to experiment several times, they rely on stable cell lines. On the other hand, the transient process is used to produce significant amounts of protein within a couple of weeks.
2. Through insects
Insects can be used for reliable protein expression processes. However, some modifications are required before initiating the process to conduct protein expression in insects. The protein expression process in insects starts with the development of recombinant baculovirus.
The produced recombinant baculovirus can then be used to produce the desired protein. Insects are preferred for protein expression as they can be optimized to create high-density suspension culture, which results in higher yields of proteins that can go up to 500 mg/L.
3. Through yeasts
Protein expression through yeasts lies under the category of eukaryotic protein expression. The stability of yeast protein expression is higher, and it has comparatively simple media requirements. It’s easier to find yeasts for protein expression. For example, t ressei is a yeast that can be used for protein expression.
The most common applications of yeast protein analysis are:
- Structural analysis
- Antibody generation
- Functional analysis
- Protein interactions
4. Through bacteria
Bacterial protein expression is more famous than other protein expression processes because it’s easier to culture cells in lab conditions. Bacteria also grow faster and can easily produce higher yields of recombinant proteins.
One area where bacterial protein expression lacks is the production of multi-domain protein expression. Simple bacteria lack the post-translational modifications which are required to produce functional proteins.
5. Through algae
Algae can also be used for protein expression purposes. The reason why algae are picked for protein expression is that it provides superb experimental control. It’s easier to optimize algae for selection and expression of proteins.
Some disadvantages come with choosing algae for protein expression. For example, algae protein expression is still a nascent technology, and it is underdeveloped from other host platforms available in the market.
The general purposes of using algae for protein expression include:
- Study of photosynthesis
- Understanding plant biology
- Study of lipid metabolism
- Genetic engineering
- Production of biofuels