Monday, November 9, 2009
P19

SCREENING for high protein secretion in mutant strains of Saccharomyces cerevisiae

Eva A. Palmqvist1, Peter Becker1, and Per Nørgaard2. (1) Novo Nordisk A/S, Cell Culture Technology, Novo Nordisk Park 1, Måløv, 2760, Denmark, (2) Novo Nordisk A/S, Protein Expression, Novo Nordisk Park 1, Måløv, 2760, Denmark

Techniques such as random- and site directed mutagenisis, and strain hybridisation enables production of a vast number of mutant strains. A challenging and important task is to identify strains within the population with the desired properties. In order to reduce the number of strains for further analysis, it is necessary to establish relevant screening systems and screening criteria. We have investigated the performance of recombinant strains of S. cerevisiae expressing glucagon like peptide-1 (GLP-1) analogues during scale-up. Strains which exhibited high secretory capacity at µmax in 5 ml and shake-flask cultures were selected for further analysis. The ability to secrete protein at a lower growth rate and under glucose limitation was then investigated in small-scale fed-batch culture, and eventually the best strains were investigated in continuous culture. The maximal productivities and critical dilution rates of the strains were investigated by automatically increasing the substrate feed-rate in continuous culture until the ethanol concentration reached a set-point value (0,150 g/l). We present data stressing the fact that cells growing under glucose limitation (production environment) and cells growing in batch culture exhibit strain dependent differences in protein productivity. The ranking of strains evaluated in batch culture can therefore be quite different from the ranking in fed-batch and continuous culture, and this has to be considered early in the screening process. The results are discussed in relation to growth-rate and physiological state of the cells.

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