Heterologous expression refers to the expression of a gene or part of a gene in a host organism that does not naturally have the gene or gene fragment in question. Insertion of the gene in the heterologous host is performed by recombinant DNA technology. The purpose of heterologous expression is often to determine the effects of mutations and differential interactions on protein function. It provides an easy path to efficiently express and experiment with combinations of genes and mutants that do not naturally occur.
Depending on the duration of recombination in the host genome, two types of heterologous expression are available, long-term (stable) and short-term (transient). Long-term is a potentially permanent integration into the gene and short-term is a temporary modification that lasts for 1 to 3 days.
After being inserted in the host, the gene may be integrated into the host DNA, causing permanent expression, or not integrated, causing transient expression. Heterologous expression can be done in many types of host organisms. The host organism can be a bacterium, yeast, mammalian cell, or plant cell. This host is called the "expression system". Homologous expression, on the other hand, refers to the overexpression of a gene in a system from where it originates.
Methods
Techniques to isolate specific genes Gene identification can be accomplished using computer-based methods known as heterologous screening techniques. A digital library of cDNA sequences has data from many sequencing projects and allows for easy access to sequence information for known genes.
If a genomic sequence is unknown or unavailable, DNA undergoes a process of random fragmentation, cloning, and screening to determine its phenotype. Although various methods can be used to obtain a particular gene, the easiest way to reveal the components of an unknown DNA sequence is by first identifying its restriction enzymes. Restriction enzymes are enzymes responsible for cleaving DNA into fragments at a specific site within molecules known as restriction sites. These enzymes can be located in bacteria or archaea and are known to protect DNA from foreign invasion of viruses. Restriction enzymes are distinct, and each recognizes only a specific sequence of base pairs within DNA, many of which tend to be palindromic. By locating each enzyme, the sequence associated with the restriction enzyme can be identified and isolated.
If the sequence is known, a technique referred to as the Polymerase chain reaction (PCR) can be used to isolate a gene of interest. The purpose of PCR is to not only identify but to amplify a particular DNA segment through phases of denaturation, annealing, and extension. Denaturation places a double-stranded DNA template in high-temperature conditions of 95 °C to break its weak hydrogen bonds and enforce strand separation. Annealing cools down the reaction to allow hydrogen bonds to reform and promote primer binding to their complementary sequences on the single-stranded template of DNA. Finally, the extension step involves DNA polymerase recognizing the primed single-stranded DNA, and therefore isolating specific sequences necessary for replication.
Techniques to incorporate genes into the host
Gene gun delivery and biolistics Gene gun delivery/Biolistics has been an attractive method for gene delivery due to its non-viral properties, and in addition to viral transduction, is one of the most common methods. This allows for less adverse immune responses and a smaller chance of viral infection compared to viral-based transfer methods. Rather than using a viral vector, this technique utilizes physical methods, specifically using helium propulsion to deliver transformation vectors. Gene gun delivery has been traditionally used for the generation of transgenic plants as it has been able to efficiently and effectively penetrate the cell walls. More recently, this technique has been successful in animal cells that cannot tolerate high-level bombardment, where instead DNA gold particles are delivered at lower helium pressure. This method has been successfully used both in vitro and in vivo.
Electroporation Electroporation is a method that uses high voltage to create pores in the membranes of mammalian cells. By pulsing with electricity, local areas of the cell membrane transiently destabilize and DNA can then enter the cell. At appropriate field strengths, damage to the host cell in minimal. This technique can be used for both short-term and long-term transfectants. It is also effective with almost any tissue type and has displayed high levels of gene delivery with an increase in the distribution of cells expressing the DNA.
Viral transduction Viral transduction is a method that uses viral vectors and is used for the stable introduction of genes into the target cells. In this method, the viral vector (virion) infects host cells that by directly transporting DNA into the nucleus of the cell. Two common types of viruses used for transduction are adenoviruses, which tend to be transient, and lentiviruses, which integrate the DNA into the genome. Lentiviral vectors have also been an attractive viral tool because they can transduce in non-dividing cells, allowing for stable transfer in a large range of host cell types.
Lipofection In lipofection, the gene is injected with the help of liposomes. The DNA sequence is encapsulated in a liposome with the same composition as the cell membrane. This method allows it to directly fuse with the membrane, or be endocytosed, which then releases the DNA into the cell. Lipofection is often used because it works with many different cell types, is highly reproducible, and is a fast method for both stable and transient expression.
Host systems Genes are subjected to heterologous expression often to study specific protein interactions. E. coli, yeast (S. cerevisiae, P. pastoris), immortalized mammalian cells, and amphibian oocytes (i.e. unfertilized eggs) are commonly for studies that require heterologous expression. In choosing a particular system, economic and qualitative aspects have to be considered. Prokaryotic expression is widely used in recombinant DNA technology to form easily manipulated proteins by well-known genetic methods with a low costing medium. Some limitations include intracellular accumulation of heterologous proteins, improper folding of the peptide, lack of post-transcriptional modifications, the potential for product degradation due to traces of protease impurities, and production of endotoxin.
Prokaryotic and eukaryotic systems, most commonly bacteria, yeast, insects, and mammalian cells, and occasionally amphibians, fungi, and protists are used for studies that require heterologous expression. Bacteria, especially E. coli, yeast (S. cerevisiae, P. pastoris), insects, and amphibian (oocyte) cells have been used as effective hosts for expressing foreign proteins. Generally, prokaryotes are easier to work with and better understood and are often the preferable host system. It is widely used in recombinant DNA technology to form easily manipulated proteins by well-known genetic methods with a low costing medium. For membrane proteins though, researchers have observed that mammalian cells are more effective. This is because there is a lack of post-transcriptional modifications in prokaryotic systems. Limitations include intracellular accumulation of heterologous proteins, improper folding of the peptide, the potential for product degradation due to trace of protease impurities, and production of endotoxin.
Escherichia coli A popular system utilized is Escherichia coli because of its rapid growth rate (~20–30 minutes), capacity for continuous fermentation and relatively low cost. Additionally, yeast has the capacity to express a high relative volume of heterologous protein. Specifically, up to 30% of proteins produced in yeast can be the heterologous gene product. There also are safe strains of E. coli that have been successfully generated to scale up production. In addition to E. coli's attractive host properties, this host is incredibly popular due to researchers having a large amount of knowledge about its genetics, including the complete genomic sequence. However, issues arise either due to the sequence of the gene of interest and those that are due to the limitations of E. coli as a host. For example, proteins expressed in large amounts in E.coli tend to precipitate and aggregate, which then requires another denaturation, renaturation recovery method. Finally, E. coli is only optimally effective in specific conditions dependent on the gene being inserted.
Bacillus subtilis Bacillus subtilis (B. subtilis) is a gram-positive, non-pathogenic organism that does not produce lipopolysaccharides (LPS). LPS, found in gram negative bacteria, is known to cause many degenerative disorders in humans and animals and affects the production of proteins in E. coli. Therefore, although it is deemed potentially safe, B. subtilis has not been officially categorized as generally regarded by the FDA as safe (GRAS). B. subtilis has genetic characteristics that readily transform it with bacteriophages and plasmids. Additionally, it can facilitate more purification steps through direct secretion into the culture medium, and can easily be scaled up because of its ability to non-specifically secrete these proteins. To date, B. subtilis has been used to successfully study different biological mechanisms including metabolism, gene regulation, differentiation, and protein expression and generation of bioactive products. It is also the most well studied gram-positive bacteria in the world, with the genomic information being widely available. Drawbacks of this host system include reduced or non-expression of the protein of interest and production of degradative extracellular proteases that target heterologous proteins. Finally, despite B. subtilis' attractive properties, these limitations result in E.coli being the default host system over B. subtilis. However, with more research and optimization, B. subtilis has the potential to produce membrane proteins in large scales.
Yeast Eukaryotic cells can be used as an alternative to prokaryotic expression of proteins intended for therapeutic use. Yeast is a single cell fungus that uses high expression levels, fast growth, and inexpensive maintenance, similar to prokaryotic systems. Because yeast is a food organism, it is also favorable for the production of pharmaceutical products, as opposed to E. coli which may contain toxins. Yeast also has a relatively quick growth rate, with a doubling time of 90 minutes on simple media, and is easily manipulated. Similar to E.coli, yeast also has the complete genomic sequence available. The most commonly used yeast is S. cerevisiae, which can carry out post-translational modifications such as protein processing and protein folding. S. cerevisiae, P. pastoris are simple eukaryotic organisms that grow quickly and are highly adaptable. Eukaryotic systems have human applications and successfully made vaccines for hepatitis B and Hantavirus. There is a progressive increase in the use of mammalian cells for recombinant technology and synthesis of complete biological activity. This system secretes and glycosylates proteins, while introducing proper protein folding and post-translational modifications. However, when increased glycosylation abilities are employed, hyper-mannosylation, or the addition of a large number of mannose, is often observed. This hinders proper protein folding. Overall, yeast is a compromise between bacterial and mammalian cells, and remains a popular host system. The cost of production for when using yeast as an expression system is high because of the slow growth and expensive nutrient requirement.
Insects