Development of a general system for the production of controlled levels of proteins in eukaryotic cells

The ability to express specifically designed proteins in the cells of higher organisms is a vital tool in modern biology and biotechnology. It is widely used to produce new medicines and vaccines for medical applications. Recent examples of this include the production of the anti-breast cancer drug, Herceptin, and the anti-human papilloma virus vaccine, Gardisil, which are produced in mammalian and insect cells, respectively. As part of a research programme aimed at producing vaccines at low cost in plants, we have developed a very efficient method for producing significant quantities of specific proteins in leaves. This method will be very useful for screening different proteins for their ability to be developed into new vaccines, especially since the proteins can be produced in only a few days. In addition, with colleagues, we have been able to show that we can use the system to express active enzymes in plants. This means we can alter the plant's metabolism so that it can produce new compounds which maybe of use to industry or medicine. In the current application, we propose to further develop our system for expressing proteins so that it can be more easily used by other researchers. A great advantage of our new system is that it is possible to control the amount of each protein that is produced. This is important when several proteins operate together since they are not always present in the same amount. Example of this are antibodies which are secreted by the skin; these consist of four different proteins which are present in different amounts in the assembled complex. Such antibodies have attracted much attention as therapies are very difficult to make by existing methods. Another use will be the introduction of a series of enzymes which can catalyse a series of reactions in a metabolic pathway. In such pathways, the enzymes are often present in very different amounts - a fact which is very important in ensuring that the correct compounds are made. Therefore being able to control the levels of each enzyme will be very important. Though originally developed as a means of the rapid production of proteins in plants, recent experiments have suggested that our system may also work well in insect cells of other higher organisms. We wish to examine if this is really true and to test expression in the cells of other higher organisms. If these experiments work as we expect, the research will have profound effects on how medicines and other important proteins are produced.

As a result of our plant expression studies, we have developed a protein expression system which has the potential to be developed into a universal, controllable eukaryotic system. By flanking a target sequence with the 5' and 3' UTRs from Cowpea mosaic virus (CPMV) RNA-2, consistent protein expression levels in plants can be achieved when the constructs are agro-infiltrated into plants in the presence of a suppressor of silencing. Furthermore, the levels of expression can be controlled by altering the sequence of the 5' UTR. The delRNA-2 system has been used to express proteins of pharmaceutical interest in plants, including ones which have proved problematic in other expression systems. In addition it has been used for the in planta expression of enzymes involved in secondary metabolism. The aim of the current application is to develop the delRNA-2 system into a system for the controlled expression of proteins in eukaryotes. We will make a number of critical improvements to the current system: (1) To enable the high-throughput expression screening of libraries, we will make the system GatewayTM - and In-FusionTM- compatible. (2) To express protein complexes and sequential steps in metabolic pathways, it is essential to co-express multiple proteins in different amounts in the same cell. We will therefore construct a variety of delRNA-2 cassettes which give varied levels of protein expression through alterations in the 5' UTR. (3) To date, all delRNA-2 plant expression studies have concerned Nicotiana benthamiana. We will investigate whether the system can be used in other plant species, such as Arabidopsis (4) While delRNA-2 has proved effective for transient expression, there are certain applications for which stable genetic transformation will be required. We will therefore transform N. benthamiana with delRNA-2 constructs expressing GFP (5) We will test whether delRNA-2 can operate in other eukaryotes by examining its expression properties in insect cells.