A high-throughput protein expression facility for structural and cell biology

While our genes provide the blueprint for life, it is the protein 'machines' that they encode that actually perform the myriad tasks necessary for the survival of humans and other organisms. For example the protein haemoglobin in the red blood cell carries oxygen from the lungs to muscles where, with the help of proteins known as enzymes, it serves to extract useful energy from sugars and other nutrients. This energy, in the form of the small molecule known as adenosine triphosphate or 'ATP', is then utilised by proteins such as myosin, termed 'molecular motors', to perform muscle contraction and other mechanical work. Other proteins, known as growth factors, convey signals from one cell to another, where they pass on information by binding to yet more proteins, the growth factor receptors, which are embedded in the membrane surrounding the cell. Upon receipt of this information, the receptors orchestrate the complex events responsible, for example, for the growth and organisation of blood vessels. Similar systems allow bacteria to sense light, oxygen and nutrients in their environments and respond appropriately. Understanding how these protein machines work is clearly of fundamental importance in biology. It also has practical importance because many such proteins are current or potentially future targets for therapeutic drugs. Similarly enzymes can potentially be 'engineered' to take on favourable properties, enabling their use for conversion of feed stocks to valuable products such as pharmaceuticals. Unfortunately, while it is relatively easy to obtain and study the genes that encode them, isolation of the proteins themselves is more difficult and currently forms a bottleneck in programmes aimed at their study. The objective of the proposed research is to overcome this bottleneck by establishing an automated facility for the rapid production of such proteins in sufficiently large quantities to enable their structures and functions to be studied in detail. This will involve introduction of the genetic blueprint for each protein of interest into a suitable host, such as the bacterium Escherichia coli. One part of the facility will comprise a robot, which will speed up the isolation of such blueprints and their introduction into the host. The bacteria can then be cultured on a large scale, in exactly the same way as yeasts are fermented for the production of alcohol. They will produce the proteins, which can then be purified using the automated equipment which is also being requested, and which can run with minimal intervention by the operator. The end result will be material suitable for detailed investigations, for example of the exact structure of the protein molecules by techniques such as x-ray crystallography and nuclear magnetic resonance spectroscopy. In combination with studies of the functional properties of the isolated molecules and how they interact with one another, important insights will be obtained into how exactly these machines help to maintain life, and how they can be exploited for pharmaceutical and industrial purposes.

Research in many fields of biology and biotechnology requires the rapid and regular production of proteins on scales up to tens of mg. Eukaryotic cDNAs and prokaryote genes are readily available from commercial and academic sources as templates. However, production and purification of the encoded purified proteins in a high-throughput and flexible fashion remains more difficult. For example, for structural proteomics projects it is typically necessary to produce, in parallel, many variants of proteins and their fragments in order to identify domains which can be produced at high level, are stable, and either susceptible to crystallisation or suitable for investigation by NMR. Similarly, high throughput production of tens or hundreds of proteins may be required for construction of protein arrays for interaction experiments, and multiple mutants of proteins may need to be produced in parallel for detailed investigation of structure/function relationships. For biotechnological applications, larger quantities of small sets of proteins may be required. Because of the vagaries of protein expression, typically multiple expression conditions, vectors and hosts need to be explored for each target to achieve reasonable levels of production. Protein production is therefore very often a bottle-neck in both structural and cell biological projects. The aim of the present proposal is to overcome this bottleneck by establishing a Facility, with robotic liquid handling and automated chromatographic capabilities, for the expression and purification of proteins. It will support multiple areas of research, including (a) enzyme mechanism and directed evolution of enzyme specificity (b) mechanisms of myosin, dynein and other molecular motors, (c) the role of viral proteins in interaction with the host cell, (d) protein complexes and protein-protein interactions and (e) mechanisms of signal transduction in mammals and bacteria.