by Amarjit Dosanjh
for Biochemistry II (CHEM 4420)
With the advent of recombinant DNA techniques, researchers have been able to produce insulin in another fashion. The human gene that encodes the information to produce insulin has been cloned into other organisms. These organisms now produce, in mass quantities, normal functional human insulin. The organism of choice, as host for such foreign genes was, for a long period of time, the bacterium, Escherichia coli. The human insulin gene has been spliced into the DNA of E. coli. This bacterial cellular factory is producing the human protein product: insulin.
Esherichia coli has been the " factory" of choice for several reasons. It is a single-celled organism that reproduces mainly through asexual reproduction. The simplicity of the organism makes it easy and cheap to work with. The food source is simple, and it does not require elaborate facilities for growth and maintenance. Its rapid growth cycle allows for a quick increase in the population size of a particular strain. An E. coli population can double in less than an hour. In addition to its own circular DNA, E. coli, as well as other bacteria, may also have an additional small segment of DNA within its cytoplasm. This additional segment is called a plasmid, and it complements the main segment of DNA. Plasmids are easy to isolate and manipulate. The plasmid can be removed, a desired gene can be inserted into it, and the plasmid can be reintroduced into E. coli. The E. coli will then produce the foreign protein as if the protein were native to the bacteria.
The protein can be of a foreign source. However, the promoter for the gene must come from the host that will be producing the protein. A promoter is the segment of DNA located immediately in front of each gene. The promoter regulates when, how much and how often the gene is transcribed. While the simplicity of E. coli makes it a desireable host for production of a foreign protein, it also has its disadvantages as a host cell. E. coli is a prokaryote. Like all prokaryotes, E. coli does not have any of the membrane bound organelles found in eukaryotes. In eukaryotes a protein is often modified after it is intially produced. Some of the best studied modications occur in different organelles, such as the endoplasmic reticulum or the Golgi apparatus. These modifications, in many cases, are necessary to convert the promoter to a functional form.
These so-called, post-translational modifications, often involve addition of different forms of glycolation. Any eukaryotic protein can be mass translated in E. coli, but many are not quite finished and hence, they are nonfunctional. E. coli will give you the same primary structure, as occurs when that protein is initially produced in its own cell type. However the failure to modify that structure often means that the protein will not form as it would with the presence of certain organelles.
Because of the handicap encountered when using E. coli to produce eukaryotic products, other organisms have been studied as suitable replacements. Mammalian, insect and yeast cells have all been studied as suitable replacements for E. coli. Of the three, yeast cells are the most desirable. They combine the ease of genetic manipulation and rapid growth characteristics a of prokaryotic organism with the subcellular machinery for performing post-translational protein modification of eukaryotic cells(4).
Many foreign proteins have been succesfully mass produced in the yeast Saccharomyces cerevisiae. This particular species is popular largely because it was familiar to molecular biologists. There is a large amount of knowledge that had been accumulated about its genetics and physiology. While this species has been used for the production of some eukaryotic foreign proteins, it has several limitations. Generally, the product yields are low. Yields reach a maximum of 1-5 percent of the total protein. The presence of foreign gene products puts additional stress on the cells. The production of the protein during the growth phase hinders growth. Even the use of inducible plasmid promoters to achieve a partial separation between the growth and protein production phase, has not been effective due to the instability of plasmid(1). Instability is especially high when the foreign protein product is somehow toxic to the yeast. In addition to the difficulties with scaling up protein production to get better yields, several reports have noted the hyperglycosylation of secreted glycoproteins which may cause differences in immunogenicity, diminished activity, and decreased serum retention of the foreign protein. Also, many of the secreted proteins of S. cerevisiae are not found free in the medium, but rather in the periplasmic space. This leads to problems with purification and further decreases product yield(1). Due to these problems mentioned above several other species of yeast have been analyzed.
One of the alternative species that has been looked at is Pichia pastoris. There are several reasons that this particular species is appealing. The protocols for its growth did not have to be worked out because it can be grown under conditions that are similar to Saccharomyces cerevisiae. Pichia pastoris has a strong, inducible promoter that can be used for protein production. It is capable of generating post-translational modifications that are more similar to human protein modifications than S. cerevisiae was capable of doing. Isolation of foreign protein is facilitated by the fact that P. pastoris does not secrete a lot of its own proteins. The first reason for its appeal is self explanatory, the latter three reasons are the ones that will be examined in this paper.
One of the drawbacks with Saccharomyces cerevisiae was that it did not have a strong inducible promoter. Pichia pastoris has a strong inducible promoter. This inducible promoter is related to the fact that Pichia pastoris is a methyltropic yeast. The first step in the utilization of methanol is the oxidation of methanol to formaldehyde and hydrogen peroxide(8). This step is catalyzed by the enzyme alcohol oxidase. The expression of this gene is tightly regulated. When the yeast are grown on glucose or ethanol, alcohol oxidase is not detectable in the cells. However, when the yeast are grown on methanol, alcohol oxidase can make up to thirty-five percent of the total cellular protein. The control of the amount of alcohol oxidase is largely transcriptional(2).
There are two alcohol oxidase genes: AOX1 and AOX2. The protein coding regions of the genes are largely homologous, 92 percent and 97 percent at the nucleotide and amino acid sequence levels respectively(9). The promoters share very little homology. No mRNA of the two genes is detectable when the yeast are grown in glycerol. The promoter region for AOX2 has a repressor region the leads to the inhibition of gene expression, and an activation region that leads to the enhancement of gene expression. The AOX1 gene promoter probably has a similar mechanism(9).
The key enzymes for methanol metabolism are compartmentalized in peroxisomes. The proliferation of peroxisomes is a reflection of environmental conditions. When the cells are grown on glucose very few peroxisomes are present. When grown on methanol, peroxisomes may take up to 80 percent of the total cell volume. Previous results clearly show that the alcohol oxidase promoter is both tightly regulated and is a strong promoter. The production of foreign protein can be repressed until the culture is saturated with colonies, and then the production of the foreign protein can begin with the derepression and induction of the gene. In addition to being able to regulate the production of the protein very tightly, the post- translational modifications made by Pichia pastoris are more suitable for use in humans. The structure of carbohydrate added to secreted proteins is known to be very organism specific. Many proteins secreted from S. cerevisiae have been demonstrated to be antigenic when introduced into mammals thus, the use of glycoprotein products synthesized by yeast for therapeutic purposes has been avoided. A comparison of a S. cerevisiae protein secreted from S. cerevisiae and P. pastoris has shown distinct differences between N-linked oligosaccharide structures added to proteins secreted from these yeast. The majority of the N-linked oligosaccharide chains are high mannose. However, the length of the carbohydrates chains is much shorter in P. pastoris. Even the longest chains of protein produced in P. pastoris contained only approximately thirty mannose residues, which is significantly shorter than the 50 to 150 mannose residue chains typically found on S. cerevisiae glycoproteins. The second major significant difference between the glycolation by S. cerevisiae and P. pastoris is that glycans from P. pastoris don't have alpha 1,3-linked mannose residues that are characteristic of S. cerevisiae(4). The enzyme that makes alpha 1,3 linkages is alpha 1,3 mannosyl transferase and it is undetectable in P. pastoris. It is significant, because the alpha 1,3 linkages on S. cerevisiae glycans are primarily responsible for the highly antigenic nature of glycoproteins used for therapeutic products(4)
The purpose of mass producing proteins is to purify them and then use them to treat diseases. One of the first steps is the isolation and purification of a foreign protein product. P. pastoris grows on a simple mineral media and does not secrete high amounts of endogenous protein. Therefore the heterologous protein secreted into the culture is relatively pure and purification is easier to accomplish(6). Secretion of the foreign protein is accomplished by recombining a signal sequence in front of the desired foreign gene when it is inserted into the host DNA.
The production of proteins in foreign hosts is an integral part of treating certain diseases that result due to a deficiency in a particular protein. While E. coli was the original host organism of choice, it has somewhat fallen out of favor due to its inability to make post-translational modifications. The original alternative was a species of yeast: S. cerevisiae. However, due to the limitations on S. cerevisiae, other species have been investigated. One of those species is Pichia pastoris. Methods for its growth were well established. It has a strong, inducible promoter. The post-translational modifications that it makes may be more favorable than those of S. cerevisiae. In addition to the above, this species does not secrete a lot of endogenous protein, so it is easier to isolate heterologous proteins. The above discussion clearly shows that there are many benfits to using Pichia pastoris as a host system for synthesizing foreign protein products.
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