- Author/Lin Jiayang|Master student in the Department of Entomology, National Taiwan University.
- Author/Lin Yuchun｜PhD student in the Department of Entomology, National Taiwan University.
- Author/Wu Yuelong｜Associate Professor, Department of Entomology, National Taiwan University.
- Author/Huang Rongnan｜Professor of the Department of Entomology, National Taiwan University.
Take Home Message
In order to produce the protein we want on a large scale, to manufacture vaccines, protein drugs, skin care products and other products, there are many ways to perform protein expression. The insect expression system has a low cost in cell culture, and its post-translational modification system is relatively close to eukaryotes, giving it advantages in production, but it is still difficult to supply a large amount of market demand.
If you can directly treat insects as a factory for protein production, use viruses to infect the insects, and then recover the protein from the body fluids of the insects, you can increase production and reduce production costs. This is also the concept of the so-called “biological factory.”
The severe cold winter is often a carnival where many viruses are raging joyously. Cunningly, they take advantage of the cold weather and people’s immunity to decline, and cause symptoms such as fever, cough, and runny nose in the human body. In order to avoid such a situation, many people choose to administer vaccines against these viruses, but how can we quickly produce so many vaccines to meet all the needs?
Traditional vaccines are mostly composed of protein, and the demand is extremely high. Therefore, there is a need for a protein production model that can be produced on a large scale, with high efficiency, and at a low cost. This production model can also be applied to other protein-related products, such as protein drugs or skin care products, but what kind of production Can the model meet so many requirements?
What methods can make the desired protein?
The first thing you think of may be genetically modified organisms (GMOs, referred to as genetically modified organisms). As long as the genetic sequence of the organism is modified, it can produce the protein we need. Although this is a feasible method, it is not feasible from the point of view of large-scale production, because the creation of genetically modified organisms cannot be achieved overnight, and it is also very difficult from the perspective of a complete biological individual level, and it also involves organisms. Various biological systems in the body.
Perhaps the modified gene will have a negative impact on organisms; or the modified gene sequence has poor performance and cannot be industrialized and mass-produced. These potential factors may make the modified gene sequence difficult and unpredictable. In addition, the cost of creating genetically modified organisms is high. In terms of the performance of specific proteins, the time and cost required to invest in genetically modified organisms is not effective.
With the advancement of science and technology, we now have more options for protein expression, including E. coli expression system, yeast expression system, insect expression system, mammalian expression system, etc. (Figure 1). Due to the differences in post-translational modification between eukaryote and prokaryote, most of the expression systems of E. coli and yeast are applied to the protein expression of prokaryotes; The performance systems of insects and mammals are applied to protein expression of eukaryotes. Although yeasts are eukaryotes, their post-translational modification systems are similar to those of prokaryotes, so most of them are still used for protein expression in prokaryotes. So how do we choose these expression systems?
Figure 1: Four different protein expression systems
From a human point of view, since humans are eukaryotes, we should choose either an insect performance system or a mammalian performance system. These two expression systems are superior to insects. Considering large-scale production, the cost of insect expression systems in cell culture is much lower than that of mammals. Mammalian cell culture requires a lot of sophisticated and expensive instruments and consumables, such as incubators and cell culture fluids that can control temperature and carbon dioxide concentration, making the conditions for cultivating mammalian cells very stringent.
Due to the high cost of the production process, if you want to use the mammalian expression system to produce protein in large quantities, there will be a certain degree of difficulty, not to mention that most of the mammalian system, whether it is at the cell level or individual organisms, has regulations. Problems, these factors create the advantage of insect performance system in production.
Use “virus” to make the protein we want!
That being the case, how can insects be used to produce specific proteins in large quantities? The answer is actually very simple, it is a “virus.”
After the virus infects the host cell, it can use the resources in the host cell to express its genes. Therefore, as long as part of the sequence of the virus gene is modified into the target protein sequence we want, the virus can produce a specific protein. Therefore, “which virus to choose” is the key to the insect expression system. This virus must have the ability to express a large number of specific genes, and its host cells must be easy to cultivate, so that the yield can be maximized. Among so many kinds of viruses, only the “baculovirus” (baculovirus) can meet so many conditions.
Currently, the most widely used baculovirus in insect expression systems is the California Alfalfa Nuclear Polyhedrosis Virus (Autographa californica Multiple nucleopolyhedrovirus, AcMNPV), a member of the baculovirus family, is a double-stranded DNA virus. Nucleopolyhedrovirus (NPV) can infect a variety of Lepidoptera (Lepidoptera) larvae, such as butterflies, moth larvae, and after the virus infects the insect body, it will produce a structure composed of “polyhedrin”, called “Inclusion body” (inclusion body).
We can imagine the inclusion body as a hard shell used to wrap the virus, which not only protects the virus from external harm, but also allows the virus to survive in the natural environment for more than one year. These capabilities make it a biological pesticide with potential for prevention and control, which can control pests without using chemical pesticides. The infection pathway of nuclear polyhedrosis virus starts from the mouthparts of lepidopteran larvae. When the larvae feed on the leaves containing the inclusion body virus, the inclusion bodies will follow the esophagus of the larvae and enter their body. The polyhedrin structure is broken down by the alkaline intestinal fluid in the larva’s midgut, and virus particles are released.
Because the virus particles are very small, with a diameter of only 250-300 nanometers (nm) and a width of only 30-60 nanometers, they can directly pass through the peritrophic membrane of the larval midgut.[Note]; When the virus particles pass through the periphagus, they will come into contact with epithelial cells in the midgut and undergo membrane fusion. At this time, the protein coat of the virus will be broken down in the cell, and the viral DNA will be sent into the cell nucleus, and begin to produce budded viruses. These budded viruses will enter the blood after the cell is lysed. In the hemocoel, other tissues and cells are further infected, causing secondary infection (Figure 2). In the late stage of infection, the virus will express a large number of polyhedrin proteins and assemble them into inclusion bodies, which are released into the environment after the worms die. Therefore, the polyhedrin structure is the key to the insect expression system.
Figure 2: The mechanism of nuclear polyhedrosis virus infection
How to make the target protein?
Since the polyhedrin protein will be expressed in large quantities in the late stage of virus infection, as long as the polyhedrin sequence is replaced with the target protein sequence, the nuclear polyhedrin virus can be used to produce the protein we want in large quantities. The concept sounds very simple, but the steps are a bit complicated.
First of all, we need to prepare a cloning vector and a transfer vector. The replication vector contains the protein gene sequence we want to insert, and it is used by polymerase chain reaction (PCR) or large intestine Bacillus culture is used to replicate in large quantities; the transfer vector contains a polyhedrin promoter (polyhedrin promoter) and a foreign gene insertion site (multi-cloning site). Then, by using restriction enzymes and ligases, we can insert the target gene sequence into the transfer vector to form a recombinant transfer vector.
The second step is to prepare to remove the treated, linear baculovirus DNA. Since the removed viral DNA is inactive, it will not affect the next step of transfection of cells; in addition, because they may also be taken up by cells, infect and produce wild-type viruses that do not contain the target sequence, so The removal of virus activity first is very important to the success rate of the experiment.
The last step is transfection. The baculovirus DNA and recombinant transfer vector are added to the cultured cells together. Since the recombinant transfer vector contains the homologous genes of the baculovirus, when the cell takes in the recombinant transfer vector and the baculovirus DNA, the two will undergo homologous recombination, making the transfer vector contain The homologous fragments of the target sequence are exchanged with the homologous positions on the baculovirus DNA to form a baculovirus containing the target sequence, which is a recombinant baculovirus (recombinant baculovirus). These recombinant baculoviruses will begin to infect cells and show the protein we want (Figure 3).
Figure 3: The steps of baculovirus expression protein
❶Prepare the replication vector and the transfer vector. The replication vector contains the target gene sequence to be inserted, and then use polymerase chain reaction or E. coli culture for mass replication; the transfer vector contains the polyhedrin promoter and the insertion position of the foreign gene. Afterwards, restriction enzymes and ligases are used to insert the target gene sequence into the transfer vector to form a recombinant transfer vector.
❷Prepare to excise the linear baculovirus DNA.
❸Add the baculovirus DNA and the recombinant transfer vector to the cultured cells. Since the recombination transfer vector contains the baculovirus homologous gene, when the cell takes in the recombination transfer vector and baculovirus DNA, the two will undergo homologous recombination, so that the transfer vector contains the homologous fragment of the target sequence. The homologous positions on the DNA of the baculovirus are exchanged to form a baculovirus containing the target sequence, that is, a recombinant baculovirus. These recombinant baculoviruses will begin to infect cells and show the protein we want.
Think of insects as “biological factories”?
In terms of the current development of baculovirus, regardless of the production cost, speed, or difficulty of establishing a production line, it has many advantages over mammalian performance systems. However, it is still difficult to supply a large amount of market demand. Using insect cells to produce protein still has a certain cost. It is not easy to expand the cell culture system. What if we use whole insects for protein production? After the virus is used to infect the worms, these proteins are recovered from the body fluids of the worms. Can this increase the yield and reduce the production cost at the same time?
In fact, this is the concept of the so-called “biological factory”, which directly treats insects as our protein-producing factories (Figure 4). Compared with cells, insects are simpler in culture and easier to operate. Unlike cells, which require special attention to contamination, there is no need for sterile operation tables, cell culture fluids, incubators, and fetal cells that provide cell growth factors. Expensive equipment and consumables such as bovine serum (fetal bovine sera, FBS). For the host of lepidopteran larvae, only air-permeable plastic containers and artificial feed are needed for cultivation, and the cost of these things is much lower than what the cells need. In addition, the number of cells in a single larva is far more than one. Cell culture dishes have made the concept of biological factories a new direction for insect performance systems.
In terms of proteins needed by eukaryotes or humans, insect performance systems have advantages that many mammalian performance systems do not have. The method of infecting insect hosts with baculovirus to express specific proteins is not only low in cost, high in yield, and not subject to regulatory restrictions, but also easier to design an industrial supply chain. If there is a demand for eukaryotic proteins, you may wish to Consider taking a look at the insect performance system!
Figure 4: Using insects as biological factories
- [Note 1]Periphagus is a membranous structure in the midgut of insects that protects the cells of the intestinal wall, just like the mucosa in the human intestines and stomach.
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