HORT 250 - Biotechnology in Agriculture

Lecture 8 - Using microbes to produce specific proteins

The ability to transform microbes (bacteria and yeasts) has made it possible to use these organinsms to produce large amounts of specific proteins, proteins that are of high value for use in medicine, agriculture and other processing activities. In this lecture I will describe how proteins can be produced in this way, and some of the applications of this technology

What is needed in order to synthesise large amounts of a protein in microbes?

1. Need to know the sequence of amino acids in the protein

• this can be obtained directly by sequenceing the protein
• more typically it is now deduced from the sequence of bases in the cloned gene and by translating these into amino acids using the universal genetic code

2. Obtain the sequence of DNA that encodes the protein

• this can be obtained from the cloned gene, but with many eukaryote genes this gene sequence also contains introns, which do not encode the protein but are instead removed from the RNA transcript before the mature messenger RNA leaves the nucleus. Bacteria are unable to remove introns from an RNA, so a gene containing introns could not be translated into the correct protein.
• a copy of the gene without introns can be obtained from a cDNA. Using the RNA as template, a complementary DNA (cDNA) molecule is synthesised. The sequence of bases in the RNA is copied into the complementary sequence of bases in the cDNA by an enzyme known as reverse transcriptase
• the correct sequence of bases in a DNA molecule can also be synthesised by a machine. The operator types in the desired sequence and the machine will assemble the desired DNA. In this method, the machines can only produce DNA molecules of 100 or 200 bases, so synthetic genes are assembled from smaller blocks.

3. The next step is to assemble this piece of DNA encoding the protein, the open reading frame, into a functional gene. Generally such genes would be called chimeras because the are put together with components from a variety of sources. The modules that are required for expression include:

• Promoter, for binding RNA polymerase and regulating transcription. The promoter is one of the most important factors in determining the level of expression of a gene in these systems. See overhead on the level of expression of different promoters in E. coli and another bacterium, a Rhizobium species.
• Transcription initiation site, where RNA polymerase starts to make RNA
• Translation start site, where the ribosome initiates protein synthesis at the first AUG codon
• Open reading frame, encoding the protein
• Translation stop site, where the stop codon terminates translates
• Transcription stop site, signalling the RNA polymerase to stop transcribing RNA

All of these modules are encoded in the DNA. These modules are the basic units, the building blocks, that are used to manipulate and modify gene expression. They can be assembled in many different combinations. In many cases, the target open reading frame of the desired protein is placed in a plasmid with all of the other components already present.

4. The constructed plasmid vector is used to transform the host microbe

5. Tests are run to determine how much protein is produced by the transformed strain of microbe. If necessary, modifications will be made to the chimeric gene in order to improve the "yield" of the target protein. This is to some extent a process of trial and error, looking for gradual, incremental improvements in the yield of protein.

6. A fermentation procedure is developed to grow large volumes of transformed microbes. Again this must be optimized to obtain a high yield of the product. Conditions must be designed to allow expression of the target protein, usually depending on the promoter used to drive expression of the assembled chimeric gene. For example, some promoters are induced by environmental conditions, such as high temperature; others are induced by the addition of specific chemicals to the medium. In most cases, fermentation is allowed to proceed without synthesis of the target protein until the microbial cells have reached a high density, close to stationary phase. Then the promoter of the chimeric gene is induced, synthesis of the protein occurs and the microbes are harvested.

7. The protein expressed in the mirobes must then be purified from the cells. This is essential for a number of reasons:

• to remove bacterial proteins that may be toxic; e.g. in the production of Humulin, the insulin sold by Eli Lilly that is made in bacteria, the goal is to remove one contaminating protein so that it is present at less than 0.1 parts per million in the final insulin preparation.
• further processing may be required to obtain an active protein. Again using insulin as an example, this hormone is synthesised as a precursor called proinsulin. The proinsulin that is produced in bacteria must be modified by an enzyme to produce the active insulin hormone.
• finally the protein must be formulated in a manner that is suitable for the final application, e.g. preparation of insulin as an injectable solution.

There are now many examples of proteins produced in this way, especially proteins that are used in the treatment of human diseases. Below I give a small sampling of some of these protein therapies, there are more already on the market with an even larger being under development and in clinincal trials.

Protein	Application
Insulin	treatment for dibetes (the first therapy derived from genetic engineering)
Growth hormone	treatment for dwarfism
Interferon	for treatment of some cancers and, I believe, multiple sclerosis
Erythropoietin	to treat anemia by stimulating bone marrow cells to produce red blood cells
Tissue plasminogen activator (tPA)	to dissolve blood clots in victims of heart attacks and, more recently, in stroke patients; tPA activates a whole cascade of processes to break down blood clots
Hepatitis B vaccine	to vaccinate against this viral disease

What are the reasons for using microbes to produce these proteins?

1. It is either difficult or impossible to purify these proteins from a natural source. For example, human growth hormone used to be produced from pituitary glands taken from human cadavers. It takes many donors to produce sufficent hormone to treat a single patient. Because of supply limitations, the treatment was not widely available.
2. The proteins purified from microbes are much safer for the patients. When proteins are purified from donors, there is a significant risk that pathogens will not be reomved and will be passed from donor to patient. An example of this is the treatment of haemophiliacs. In the past, clotting factors used to treat these patients were purified from donated blood. Before the virus responsible for AIDS was identified, it was impossible to test donated blood for this virus, HIV. Many (perhaps most) haemophiliacs who used clotting factors became infected with HIV because these were contaminated with the virus that causes AIDS. There are many other pathogens that can be transmitted in this way. Clotting factors and growth hormones purified from microbes are very unlikely to be contaminated with human pathogens. Vaccines produced through recombinant DNA technology may be safer. For some diseases, live vaccines are still used, i.e. they contain live viruses. There is concern that these may not always be as mild a strain of the virus as believed and may in some individuals cause a severe disease. If a proetin from the virus can be expressed in microbes and used as a vaccine instead, it reduces the risk of unintentional infection.
3. In a number of cases, these potential therapeutic agents were unknown until they were discovered in the last several years. The discovery of some of these proteins was the result of advances in molecular and cellular biology.

In agriculture, one of the first uses of proteins produced in bacteria has been to increase milk production in cows. It has been known for many years that injection of bovine somatotropin (bST, BST, a growth hormone) into lactating cows could produce a significant increase in milk production. However, it was impossible to obtain sufficient BST to do this on a commercial scale. However, once the gene encoding BST had been cloned, it became possible to produce this protein hormone in bacteria. BST produced in this way is now sold by at least two companies, as Posilac by Monsanto, and also by Elanco, a division of Eli Lilly. There has been a great deal of controversy about the use of BST, the effect of this on cows, and the safety of the milk produced from treated animals. We will examine some of these issues in another class.

In summary, producing proteins in microbial fermentation systems is now widely practiced. It offers a method to produce proteins that could not otherwise be purified in sufficient quantity for practical use. The design and assembly of the chimeric genes are critical to the sucess of this approach. Of particular importance is the choice of promoter to drive expression of the gene to obtain a high level of protein synthesis. A variety of organisms can be used to produce these proteins, including bacteria and yeasts; plant or animal cells are also used in some cases. Optimizing the fermentation and purification can be vital to obtaining a sufficient yield of the target protein.

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