Protein Engineering

Tony Pembroke

Introduction

 Over the past 20 years there has been a major scientific revolution unfolding in the area of genetic engineering, that is, the ability to isolate, study and manipulate the genetic material of all living organisms. Although the origins of genetic engineering come from the study of Microbiology, this new discipline has been accepted warmly by many other scientific disciplines as a means of analysing and solving problems in many diverse areas of the life sciences. For example, at present there is a major international research initiative underway called HUGO, the Human Genome Project. Its aim is to study the genetic script of humans. This could ultimately result in a real understanding of the molecular basis of human disease and genetic disorders.

In the area of Biochemistry, the science of the study of the chemicals that make up living things, there is emerging another revolution based on genetic engineering. This is known as protein engineering and this is the ability to alter or change in a ratlonal manner the structure and/or function of proteins or to create new proteins that do not occur naturally.

This may lead to a revolution bigger than the microelectronics revolution, yet the basis of protein engineering is, as yet, not widely understood by the general public.

The Double Helix

 The fact that the chemical Deoxy Ribonucleic Acid (DNA) carries the genetic message was not elucidated until the mid 1940's, through the elegant experiments of O.T. Avery. This led to the discovery of the structure of DNA in 1952, a feat achieved by a biologist and a physicist - James Watson and Francis Crick. They demonstrated from X-ray studies that DNA consisted of a double helix, along molecules that looked like two springs wrapped around each other (Figure 1).

In fact we know today that the genetic message in humans and other living things is stored in the form of DNA in the nucleus of living cells, as long string-like materials, incredibly folded in the form of chromosomes. But how can a chemical polymer like DNA carry a message? This is a question that had puzzled biologists for many years until the proposal by Crick, which he called the central dogma: DNA makes RNA, which in turn makes protein. In other worlds, DNA is copied into another chemical form called RNA (RiboNucleic Acid) and this is translated by 'machinery' found within each and every cell, the ribosomes, into protein. As we now understand it, DNA is like a large floppy disk which contains a programme written in code. This code is transcribed into a different code by RNA, like the machine code in a computer, this is eventually transcribed in a 'readable form' which is the protein sequence. The DNA message in each cell is split up into domains called genes, each of which can encode the information for each protein.

What are Proteins?

 Structurally proteins are polymers of amino acids, which have a defined sequence which is dictated by the genetic message in DNA. Each of the 20 common amino acids which are found in proteins differ chemically from each other in size, charge, polarity and in the nature of their side groups. This means that when they polymerise i.e. join together like beads, to form a protein, then variou interactions occur between them resulting in a unique three-dimensional pattern for each protein (see Figure 2).

Proteins play a vital role in many cellular processes. Some have a structural role such as keratin in hair. Some have a protective roles such as antibodies, which protect against disease. Some act as chemical communicators like the protein hormone insulin. Some proteins have a nutritional role like casein in milk, which is passed on from cow to calf. Some have a transport role like haemoglobin, which carries oxygen around the body.

A large number of proteins have a catalytic role and are known as enzymes, which are used to catalyse biochemical synthesis or to break down molecules within each living cell. Most of these biological catalysts can be extracted and used outside the body. For instance in biological detergents to break down food stains, as degraders of starch. In fact the potential uses of protein enzymes are almost endless.

With better chemical and biological techniques of analysis, new proteins, which exist in the body in trace amounts, are continually being discovered. Examples include the wonder anti-cancer proteins, the interferons. Others include specific growth factors such as nerve growth factor which is a protein that can stimulate new nerve growth, which may have applications in regenerating dead nerve tissue.

Protein Engineering

 As we have already seen, the biochemical structure of a protein is encoded by the genetic message in DNA which specifies the protein. Each protein is encoded by a unique gene in DNA so that the many thousands of different proteins in living things are encoded by thousands of individual genes. The techniques of genetic engineering allow scientists to fish out individual genes from the thousands found in a chromosone. Once pure, this sequence can be modified or altered using a process called 'in vitro' mutagenesis, which allows one to introduce rational changes into the genetic message. Any change introduced will change the sequence of amino acids in the protein encoded, which may result in the protein encoded, which may result in the protein folding differently, altering its activity or bestowing a new activity on it. This process of introducing changes into protein molecules is termed protein engineering. In most cases the changes introduced are done by starting at the genetic level. However, changes can also be introduced via chemical modification in the purified protein using a variety of organic reactions, although such changes are not as stable as those introduced at a genetic level.

The Applications of Genetic Engineering

 There is a range of scientific and industrial applications of protein engineering. At the scientific level engineering proteins can be used to study the role of particular amino acids on the three-dimensional shape of a protein. By introducing rational changes in a defined order, one can analyse biochemically how each amino acid or group of amino acids affects the folding and shape of a protein.

This area of biochemistry is of major interest at the moment as once one understands how and why a protein folds in a particular way, one can then design proteins at will. This may result in totally synthetic proteins which have never existed before. One use of such proteins could be to bind to and degrade plastics, to help clean up our environment. Another possibility might be to construct synthetic proteins which would bind to viruses such as the AIDS virus and inactivate it. Some such projects are already under way in various biochemical laboratories. However, it may be a long time before we see the applications.

Work on protein engineering which has already been successful has involved altering the chemical properties of protein enzymes. In general most enzymes work best at an optimum pH and temperature, usually pH7 and 37ºC. However, many applications of enzymes as hydrolytic agents or in biochemical synthesis require them to work at much higher temperatures, e.g. 95ºC. The higher temperatures may overcome viscosity problems or make the process more economic. Recently biochemists have been able to introduce changes into many proteins which confers thermostability to the protein thus making the protein of more use and in some cases allowing the development of processes which would not be viable otherwise. At the moment the ground rules for engineering proteins are only in the preliminary stages of being examined. Future developments will, I believe, lead to huge advances in the understanding of protein structure and function, and the design of new catalytic entities.

Dr. Tony Pembroke is a lecturer at UL. His research interests are genetic engineering and Enzymology.


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Last updated 27th February 1996 by Stephen Childs