Understanding the -tech- in Biotechnology
This page is designed to introduce you to the biological molecules used in biotechnology, and guide you through the basic principles of genetic engineering. We will then take a closer look at some of the challenges involved and how these can be overcome by modern refinements.
Developing new drug therapies through biotechnological processes
You may have noticed that the recently available beta interferons are referred to as 'recombinant proteins' They are produced using biotechnological processes.
The beta interferons are included in the growing number of drugs which are produced in this way and are part of our everyday lives. Some are life-saving therapies, while others enhance the quality of life for millions of people.
Yet the same techniques that produce these vital therapies sometimes provoke anxiety because of their genetic basis. Have you wondered how ‘recombinant drugs’ are made?
A deeper understanding of the rationale behind biotechnology and its logistics might help to allay any fear or confusion by showing why such an approach is so valuable.
A definition of biotechnology
bi-o-tech-nol-o-gy
The development of techniques for the application of biological processes to the production of materials of use in medicine and industry. For example, the production of many antibiotics relies on the activity of various fungi and bacteria. Recent techniques of genetic engineering have enabled the large-scale production of hormones, vaccines, interferons, and other useful products.
Why proteins are essential
Proteins are the major structural and regulatory molecules essential for life. Some proteins may also be useful therapeutically. For example, diseases caused by a protein deficiency can be treated with the human protein itself.
Unfortunately, although the value of human proteins in treating disease has long been known, only tiny quantities can be extracted from human tissues.
With biotechnology, however, it is now possible to produce sufficient amounts of human proteins of the highest quality to use therapeutically.
The basic equation is a simple one: the DNA in the genes codes for a template called RNA , which in turn codes for proteins (see diagram). Biotechnology uses this equation on a large scale to produce therapeutic quantities of drugs.
The first steps
To understand how biotechnology can be used to make therapeutic proteins, let's first go back to 1944, when scientists discovered that genetic information is stored in deoxyribonucleic acid (DNA) in the nucleus of cells, rather than in proteins as previously believed.
This finding was a great breakthrough, because it was the first step towards the most important equation in biotechnology: genes carry a code for proteins.
In 1953, Crick and Watson showed how this could work, by deciphering the structure of DNA itself using the experimental data provided by Maurice Wilkins and Rosalind Franklin. Their work was honoured with the Nobel Prize for Medicine in 1962.
Crick and Watson showed that DNA consists of two enormously long chains of simple repeating units, entwined in a double helix. Each chain has a 'backbone' from which different rungs (bases) project inwards like a spiral ladder. There are four different bases: adenine, guanine, thymine and cytosine. For structural and electrical reasons, adenine always pairs with thymine, and cytosine with guanine.
Cracking the codon
The implications of the structure of DNA were not lost on Crick and Watson. If you ‘unzip’ the double helix, you get two single chains. Because of the way in which the bases pair with each other, each of the single chains forms a template from which an exact replica of the parent molecule can be reproduced.
This is important, because heredity depends on perfect replication. In the graphic on the below, you’ll see that this is inherent in the structure of DNA.
Crick and Watson suggested that the sequence of bases in the DNA molecule somehow codes for proteins.
There are 21 different amino acids, and these constitute the basic building blocks of all proteins. In a protein, the amino acids are joined end-to-end in a long string, which is twisted into a complex three-dimensional shape. The sequence of codons in DNA, which determines the complete sequence of amino acids in a protein, is referred to as a gene. A typical human cell has approximately 100,000 genes in its DNA.
Scientists finally cracked this genetic code in the early 1960s. They showed that the currency of genetic information is a sequence of three bases, called a codon. Each codon specifies a single amino acid.
How a protein is made
To make a protein, DNA is ‘unzipped’ along the length of a single gene. The ‘unzipping’ exposes a template on which a daughter nucleic acid, called ribonucleic acid (or RNA), can be formed by an enzyme called RNA polymerase.
This process is called transcription because genetic information is transcribed, or written in a different script (see diagram).
Once formed, RNA migrates from the nucleus to the great protein-building factories of the cytoplasm - the ribosomes - which translate the sequence of codons into the specified string of amino acids. to form a protein. You can think of this as translating a set of instructions into a different language.
From the ribosomes, newly formed proteins feed into a packaging plant - the endoplasmic reticulum - where they are clothed with carbohydrate chains to form a functional protein complex. This final packaging is called glycosylation. It has important repercussions in biotechnology, as we will see later.
The dawn of biotechnology
All cells have similar machinery for making proteins from DNA, even very simple ones like bacteria. In fact, biotechnology didn’t become possible until several important proteins (enzymes) were discovered in bacteria and viruses in the early 1970s. These include:
• restriction endonucleases (which slice up DNA at restricted sites)
• DNA ligases (which glue the DNA fragments end-to-end)
• reverse transcriptase (which reverses transcription, from RNA to DNA)
These enzymes are the tools of biotechnology. They can be used to slice up, then recombine important human genes with the DNA of bacteria.
Recombination of human and bacterial genes is the central principle of biotechnology, and gives its name to recombinant DNA technology. It is valuable because bacteria replicate very quickly. If you get the conditions right, bacteria will double in number every 20 minutes. This means they can be used to grow large amounts of protein for therapeutic purposes.
The modern synthesis
To make a recombinant protein, first isolate some human DNA and slice it up using a restriction endonuclease. This results in a lot of characteristic fragments of DNA, which contain particular genes.
Then, repeat the process with bacterial DNA, some of which is found in loops called plasmids. Usually, if you use the right slicer, you can cut the loop open in just one place.
Next, mix a fragment of human DNA, carrying the gene you need, with the opened bacterial plasmid.
Finally, recombine the human and bacterial fragments with a DNA ligase, to create a new plasmid containing the human gene.
Once you have made a recombinant DNA plasmid, you have to transplant it back into the bacteria, so it can be used to grow your protein. This technique is called transfection. Bacteria containing the human gene in their plasmids are grown in cell culture and begin to synthesise the human recombinant protein using their own cellular machinery.
Think big
Producing a recombinant protein by these methods on an industrial scale for medicinal use is technically very demanding, involving delicate handling of materials and elaborate quality control.
The major production steps are:
• fermentation - bacteria are cultured in a series of sealed vats containing a nutrient soup (culture)
• harvesting - the cells are centrifuged following fermentation to recover the protein from inside the cells
• purification - the protein of interest is isolated from approximately 5,000 normal bacterial proteins using techniques such as chemical precipitation and chromatography
• formulation - the required protein is modified to a stable, sterile form, which can be given therapeutically. Remember that proteins cannot be taken as pills because they are broken down in the stomach.
Moving from the laboratory test tube to an industrial fermenter is not always a straightforward process. Each of the production steps requires incremental manipulations to maximise the yield, purity, activity and stability of the drug.
This may be easily achieved with some compounds, requiring only a few adjustments to the process. Others are more particular and can demand much time and attention. This in turn may increase the cost of the final product. However, it will not outweigh the expense of isolating the compound from the original source in medicinal quantities, even if it is possible.
Bacteria or virus?
When producing recombinant proteins for therapeutic use, the purity, yield, biological activity and stability also depend on the type of cell used to grow a protein.
Nowadays, all kinds of different cells can be used, as well as bacteria. For example, you can use a virus to insert human genes into the DNA of mammalian or insect cells, and grow these in culture.
However, some of these cells do not grow well in culture, and most of them (including bacteria) produce slightly modified proteins that are often not as therapeutically effective as the natural human protein.
A difficult dilemma
The obvious solution to the problem of reduced activity of recombinant proteins is to grow the proteins in human cells.
Unfortunately, human cells cannot be grown under industrial fermentation conditions, and even when they are cultured on a small scale, they do not produce much protein. But there is a way out of this dilemma, and it depends on the nature of the DNA code.
Remember that a triplet of bases encodes an amino acid. If you change a single codon in DNA you can alter the amino acid sequence of the protein. This can be done using the tools of biotechnology, analogous to evolution by natural selection.
As in evolution, most structural changes to the amino acid sequence of proteins are detrimental, but sometimes a change can produce an enhanced version of the original. Sometimes, changing a single amino acid can make a protein so much better that its therapeutic activity and stability are similar to those of the natural human protein.
Looking forward
Modifying genes to produce a protein with a slightly different amino acid sequence does not sacrifice yield, because the new recombinant protein can still be grown in bacteria, which replicate faster than any other type of cell.
A whole range of modified (second-generation) recombinant proteins are already available for therapeutic use, including new forms of insulin, growth hormone, tissue plasminogen activator, factor VIII and interferon beta-1b.
This gives new hope for the future. Finally it is possible to influence the progression of diseases like MS. Now that large quantities of active therapeutic protein can be produced using the new techniques of biotechnology, effective therapies, such as beta interferon, can be brought to all those who need them.
