Expression And Identification Of Rat DAF SCR1-4

Complement activation is the basic pathological reaction in inflammatory diseases. How to control this process is a hot topic.Decay accelerating factor(DAF) is a membrane bond complement regulatory protein which could dissociate the C3, C5 convertase and prevent their formation in vivo in both classical pathway and alternative pathway. This could provide great protection to the host cells who bear these molecules. Many attempts to express soluble DAF have been made before, almost all bearing eukaryotic secretion signal peptide to facilitate secretion expression or directly in the eukaryotic cell's cytoplasm. Since DAF bears 4 short consensus repeat(SCR), which in all contain 8 pairs of disulfide bond, previous attempts to express this fragments in E.coli have ended up with inclusion bodies. In my research, I proposed to express the four consensus repeat of DAF in E.coli using a strategy of fusing this fragment to the Trx tag in the hope of obtaining soluble product.Firstly, I subcloned the DNA fragment that encode the four consensus repeat of DAF into the prokaryotic expression vector pET32a(+) which could add a Trx tag on the N-terminal of the target protein. The reconstructed vector is then transformed into the expression host BL21(DE3) pLysS.In the subsequent experiments, three parallel sets of induction conditions with different temperature and IPTG concentration were attempted. A slight increase in the soluble product was observed when the cultures were inducted...