Decay Accelerating Factor: E. Coli Expression, Refolding, Establishment Of An ELISA System And Its Metabolization In Rat

The purpose of the present study is to produce enough amount of soluble DAF for in vivo studies, i.e. the metabolization of soluble DAF in the blood and its inhibitive effect to the complement system. In order to achieve this goal, the 4 short concensus repeats (SCR1-4) of rat DAF (rDAF) and human DAF (hDAF) were successfully overexpressed in E. coli. The inclusion body was purified and refolded to obtain soluble and active DAF. Then, the recombinant rDAF was used as an immunongen to produce monoclonal antibodies against rDAF. Finally, an ELISA system was established in which as little as 10 ng/ml of rDAF could be detected. A monoclonal antibody which could detect as little as 40 ng of rDAF was isolated. The results of the in vivo experiment showed that rDAF was metabolized rapidly in the blood. Within 90 min after injection, it could not be detected. Even at the highest concentration, the negative effect of soluble DAF against the complement system was not detected. This result showed that soluble DAF is not a good candidate of complement inhibitor. In the mean time, two other studies were done. First, an efficienct and economic way of refolding DAF was established. Second, the possibility of soluble expression of DAF was explored and a possible reason was given for the soluble inclusion bodies which were formed by the NusA-DAF fusion protein.