| title: | On-line optimisation of in-vivo isotopic labelling of recombinant proteins. |
|---|---|
| reg no: | ETF6847 |
| project type: | Estonian Science Foundation research grant |
| subject: |
2.7. Biotechnology, Food and Drink Technology |
| status: | accepted |
| institution: | TTU Faculty of Science |
| head of project: | Kalju Vanatalu |
| duration: | 01.01.2006 - 31.12.2009 |
| description: | There is an increasing demand for the production of recombinant and isotopically labelled proteins and peptides both in science and biomedicine. Our group has developed several new fermentation methods which have been used also for the production and purification of many recombinant and isotopically labelled proteins. In spite of this we have to admit that the whole production process is complicated and the yields in different steps vary greatly for obscure reasons. It is known that stress mechanisms take place in (bacterial) cells when the conditions become unfavourable (eg heat shock and cold shock), stress is evoked by the synthesis of defective or of a foreign protein. Apparently also the rapid changes in the cultivation conditions can evoke the stress mechanisms in the cell which in turn can reduce the level of production of the heterologous protein. Thus, in addition to the control over stress mechanisms also the cultivation parameters have to be tuned out to achieve when optimising the protein expression. Therefore the aim of this project is to study(and later also to control) the state of the cells during the stage of cultivation/expreession. For this we intend to apply bacteria with stress-reporters and also to use (the other) fluorescent reporter for the measurement of the level of synthesis of the recombinant target protein. In order to choose the appropriate stress-protein it is planned to study the expression of different stress-proteins at variable cultivation conditions with the use of 2D-electrophoresis. The existing fermenter-computer system is capable to automatically optimise the growth of cells- to maximise the growth rate or find the best optimal ratio of different growth substrates added simultaneously. As we already have constructed the device for measuring optical density on-line we can use this experience also for the construction of on-line fluorimeters. This will enable us to follow on-line the processes occuring in the cell- the level of stress and the synthesis of the recombinant target protein. Such a system would make it possible to carry out series of optimisation experiments automatically by the computer according to the pregiven variation of cultivation parameters and also to apply control-algorithms for the automatic optimisation of the process of recombinant protein production. This approach will make it possible to better understand the intracellular processes during the production of recombinant proteins due to the on-line measurements and significantly increase theefficiency of the whole process due to the better yield, saving time and material expenditures and reducing risks. As objects of study we plan to use mainly the proteins of the hedgehog-signalling pathway: SKI, Gli-proteins, Dyrk, SUFU. |
| project group | ||||
|---|---|---|---|---|
| no | name | institution | position | |
| 1. | Risto Tanner | |||
| 2. | Katrin Tomson | |||
| 3. | Robert Tsanev | |||
| 4. | Kalju Vanatalu | Tallinna Tehnikaülikooli Matemaatika-loodusteaduskond | ||