Expression of recombinant proteins

In this article, I briefly describe the various ways of expressing recombinant proteins.

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Recombinant proteins

Proteins can be artificially produced by inserting a gene encoding the desired protein into a host organism. This process is carried out with the help of recombinant DNA technology. This way, the host cells can express and produce large amounts of protein. The first step of this process includes gene isolation and cloning, followed by transformation. The DNA sequence coding for the desired protein is first isolated and inserted into a suitable expression vector. In transformation, the recombinant vector is introduced into a host organism, such as E.coli, yeast, or mammalian cells. Transformation is followed by protein expression, which includes the culture of host cells. The cultured cells start to produce the recombinant protein. In the final purification step, the protein is extracted and purified.

Different systems express recombinant proteins

Any organism can act as a host to express proteins from a heterologous species. The organism can be bacteria, yeast, insects and insect viruses or may be mammalian cells.

Bacteria

These are the most common hosts for protein expression. E.coli is mostly used for expressing proteins as its regulatory sequences governing gene expression are well understood. Bacteria can be conveniently cultured and maintained in laboratories using cost-effective growth media. Additionally, there are efficient techniques for introducing DNA into bacteria and extracting it. Large-scale bacterial cultures can be grown in commercial fermenters, making them a valuable source of cloned proteins. However, certain foreign proteins expressed in bacteria may not undergo essential post-translational modifications or proteolytic cleavage, which are often required for their proper functionality.

A gene sequence may carry certain features, which make the expression of a particular gene difficult in bacteria. Eukaryotic proteins commonly possess intrinsically disordered regions. During their expression in bacteria, many eukaryotic proteins aggregate into insoluble cellular precipitates called inclusion bodies. This makes some eukaryotic proteins inactive when purified from bacteria. To fix this problem, new bacterial host strains with engineered eukaryotic protein chaperones or enzymes are consistently being developed.

The specialized systems exist in bacteria

Various specialized systems exist for protein expression in bacteria. Typically, the promoter and regulatory elements of the lactose operon are linked to the gene of interest to control transcription. When lactose is introduced into the growth medium, the cloned gene is transcribed. However, the lactose-based regulatory system is somewhat leaky, meaning it does not fully switch off in the absence of lactose. This can be problematic if the cloned gene produces a toxic protein for the host cells. Additionally, transcription from the Lac promoter may not be sufficiently efficient for certain applications.

The bacteriophage T7 serves as an alternative system by utilizing its promoter and RNA polymerase. When a cloned gene is linked to the T7 promoter, transcription is carried out by T7 RNA polymerase instead of the E. coli RNA polymerase. The gene encoding T7 RNA polymerase is separately introduced into the same cell within a construct that ensures strict regulation. This polymerase is highly efficient and enables strong expression of genes fused to the T7 promoter. This system has been employed for the expression of the RecA protein in bacterial cells.

Yeast – The most known eukaryotic organism

Saccharomyces cerevisiae is one of the most well-studied eukaryotic organisms and is also among the easiest to cultivate and modify in the laboratory. This yeast can also be grown on low-cost media. The tough cell wall of yeast makes it difficult to introduce DNA vectors into them. Thus, much of the genetic engineering and vector maintenance can be done conveniently in bacteria. So, the yeast vector was first propagated in bacteria. This leads to the existence of many shuttle vectors.

A protein is expressed in the same way in both yeast and bacteria. To achieve high-level expression in yeast cells, cloned genes must be linked to suitable promoters. For instance, the yeast GAL1 and GAL10 genes are regulated based on the growth medium—they are activated in the presence of galactose and repressed when grown in glucose. Therefore, if a foreign gene is expressed using these regulatory sequences, its expression can be controlled by selecting the appropriate growth medium.

Protein expression in both yeast and bacteria can present certain challenges. In yeast, foreign proteins may struggle to fold correctly. Additionally, yeast may lack the necessary enzymes to modify proteins into their active forms, or specific gene sequence characteristics might interfere with protein expression. However, since Saccharomyces cerevisiae is a eukaryote, it can sometimes express eukaryotic genes more efficiently than bacteria. Furthermore, proteins produced in yeast are often folded and modified more accurately compared to those expressed in bacterial systems.

Insect viruses- Baculoviruses

These are insect viruses, that act as parasites upon their insect larval hosts. Their genome consists of double-stranded DNA. Baculoviruses infect insect larvae, ultimately converting them into virus-producing factories. During the later stages of infection, these viruses generate large quantities of two proteins, p10 and polyhedrin. However, neither protein is essential for virus production in cultured insect cells. The genes encoding these proteins can be replaced with those for a different protein of interest. When the modified virus infects insect cells or larvae, the introduced protein is often produced at exceptionally high levels.

The baculovirus Autographa California is mostly used for protein expression. It is a multicapsid nucleopolyhedrovirus and has a large genome for direct cloning. The process of virus purification is also tedious. To resolve this problem bacmids are created. Bacmids are large circular DNAs including the whole baculovirus genome along with sequences that permit replication of the bacmid in E.coli. The gene of interest is first inserted into a smaller plasmid and then integrated into a larger plasmid through site-specific recombination within a living system (figure 1). The resulting recombinant bacmid is then extracted and introduced into insect cells. After the infection cycle is complete, the desired protein is recovered.

**Figure 1: Construction of a vector for protein expression in baculoviruses**

Viral vectors: A powerful tool for gene delivery in mammalian cells

One of the most effective ways to deliver cloned genes into mammalian cells is by using viruses. This technique takes advantage of the virus’s innate ability to transfer its genetic material—either DNA or RNA—into host cells, and sometimes even integrate it into the host genome. Scientists have developed various modified mammalian viruses to serve as vectors, including human adenoviruses and retroviruses. In this process, the target gene is inserted under the control of a viral promoter. The virus then infects the cell through its natural pathways, delivering the engineered genome, which enables the expression of the desired protein within the host cell.

In such systems, protein expression offers a significant advantage. Depending on the conditions, proteins can be produced either temporarily or continuously. When the viral DNA remains separate from the host genome and is eventually broken down, the protein expression is temporary. Conversely, if the viral DNA becomes integrated into the host cell’s genome, it enables long-term or permanent protein expression. By selecting an appropriate host cell, it is possible to achieve the correct post-translational modifications needed to activate the protein. However, culturing mammalian cells is quite costly, so this method is typically used to study a protein’s function within a living system rather than for large-scale protein production.

Conclusion

Proteins can be artificially produced using recombinant DNA technology by inserting a gene encoding the desired protein into a host organism. This involves isolating the gene, cloning it into an expression vector, and introducing the vector into a host like E. coli, yeast, or mammalian cells, enabling them to produce the protein in large amounts. After transformation, host cells are cultured to express the recombinant protein, which is then extracted and purified.

Bacteria are the most common hosts for protein expression. E. coli is a common host for protein expression due to its well-known gene regulation and ease of cultivation in low-cost media. Various specialized systems exist for protein expression in bacteria.

Saccharomyces cerevisiae is a well-studied, easy-to-cultivate eukaryote that grows on low-cost media. Due to its tough cell wall, DNA vectors are often first propagated in bacteria, leading to the use of shuttle vectors. A protein is expressed in the same way in both yeast and bacteria. To achieve high-level expression in yeast cells, cloned genes must be linked to suitable promoters.

The baculovirus Autographa California is mostly used for protein expression. It is a multicapsid nucleopolyhedrovirus and has a large genome for direct cloning. Bacmids are large circular DNAs including the whole baculovirus genome along with sequences that permit replication of the bacmid in E.coli. Viruses, like adenoviruses and retroviruses, are effective gene-delivery tools for mammalian cells due to their natural ability to transfer and integrate genetic material.