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

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

Functional mapping of the anti-idiotypic antibody anti-TS1 scFv using site-directed mutagenesis and kinetic analysis

Recombinant antibodies may be engineered to obtain improved functional properties. Functional mapping of the residues in the binding surfaces is of importance for predicting alterations needed to yield the desired properties. In this investigation, 17 single mutation mutant single-chain variable fragments (scFvs) of the anti-idiotypic antibody anti-TS1 were generated in order to functionally map amino acid residues important for the interaction with its idiotype TS1. Residues in anti-TS1 determined to be very important for the interaction were identified, Y32L, K50L, K33H and Y52H, and they were distributed adjacent to a centrally located hydrophobic area and contributed extensively to the interaction energy (≥2.5 kcal/mol) in the interaction. Quantitative ELISA assays, BIAcore technologies and three-dimensional surface analysis by modeling were employed to visualize the consequences of the mutations. The expression levels varied between 2–1,800 nM as determined by ELISA. All the 17 scFvs displayed higher dissociation rates (60–1,300 times) and all but two of them also displayed faster association rates (1.3–56 times). The decrease in affinity was determined to be 1.6–12,200 times. Two of the mutants displayed almost identical affinity with the wild-type anti-TS1, but with a change in both association and dissociation rates. The present investigation demonstrates that it is possible to generate a large panorama of anti-idiotypic antibodies and single out a few that might be of potential use for future clearing and pre-targeting purposes of idiotypic-anti-idiotypic interactions