Beschreibung:
<jats:title>Abstract</jats:title>
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<jats:title>Background</jats:title>
<jats:p>Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions.</jats:p>
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<jats:title>Results</jats:title>
<jats:p>In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-keratin 8 scFv TS1-218, during expression in <jats:italic>P. pastoris</jats:italic> in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization.</jats:p>
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<jats:title>Conclusion</jats:title>
<jats:p>The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that <jats:italic>P. pastoris</jats:italic> is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.</jats:p>
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