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Microb Cell Fact. 2011 May 16;10:34. doi: 10.1186/1475-2859-10-34.

Optimization of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using design of experiments.

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  • 1Department of Chemistry and Biomedical Sciences, Karlstad University, S-651 88 Karlstad, Sweden.

Abstract

BACKGROUND:

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.

RESULTS:

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 P. pastoris 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.

CONCLUSION:

The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.

PMID:
21575236
[PubMed - indexed for MEDLINE]
PMCID:
PMC3119184
Free PMC Article

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