| Autor: |
Rimšeliene Renata, Kraft Mario, Horn Uwe, Šiurkus Juozas, Panula-Perälä Johanna, Neubauer Peter |
| Jazyk: |
angličtina |
| Rok vydání: |
2010 |
| Předmět: |
|
| Zdroj: |
Microbial Cell Factories, Vol 9, Iss 1, p 35 (2010) |
| Druh dokumentu: |
article |
| ISSN: |
1475-2859 |
| DOI: |
10.1186/1475-2859-9-35 |
| Popis: |
Abstract Background Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale. Results Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor. Conclusions Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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