Yeast Surface Rendering Technique In Vitro Evolution Of The DAF And DAF Decay-accelerating Mechanism

To avoid excess complement activation and to protect the hosts, the complement system was tightly controlled by proteins present in the fluid phase and on the cell membranes. Decay-accelerating factor (DAF) is one of the most important complement regulatory proteins on membrane, which accelerates the decay of the classical and alternative C3/C5 convertases by inhibiting the assembly of C3/C5 convertases.It can protect cells from complement-mediated lysis and prevent the forming of anaphylatoxin (C3a and C5a), thus being a potential ideal drug that block complement activation for the treatment of several human diseaseas.Additionally, it has been found that transgenic animal organs of human DAF(CD55) could control the hyperacute rejection induced by complement activation in xenotransplantation. However, there is some defects in the properties of DAF, such as its limited decay-accelerating activity and lacking of cofactor activity of I factor, which limited its usage in clinic. It is also the receptor of some pathogens, which are potentially harmful to xenotransplantation. So reform DAF is a better choice and emergent job. We have performed the in vitro evolution of DAF to screening DAF mutant with high activity by yeast surface display on the basis of the investigation of key animo acids with functional activity in DAF.Finally we explored further the mechanism of the decay-accelerating action of DAF. The experiments consisted of six parts and the results were as follows: ? Supported by National Natural Sience Fundation(30080032) IV 1. Display of human native decay-accelerating factor on the surface of yeast The eDNA of human DAF was used as PCR templates and the yeast surface displaying vectors with mature DAF molecures were constructed by ligating with the displaying vector pYD 1, then the vectors were transformed into yeast cells and the DAF molecules were expressed on the surface of yeasts. DAF induced on the surface of yeast were labeled with three different monoclonal antibodies against DAF different epitopes and detected by fluorescence microscope, cell ELISA and FACS respectively. The result showed that three mAbs could bind onto DAF displayed on the surface of yeast. It was indicated that DAF clould be expressed on the surface of yeast and the DAF protein could be folded correctly and formed the correct conformation. This was the first report about displaying proteins except for superfamily of immunoglobulin. 2. Comparison of SCRs and selecting of mutant sites of DAF By comparing the amino acids sequences of SCRs of different regulators of complement activation (RCA) and homologous structure units of DAF in different species, the conservative and nonconservative amino acids in DAF were determined. The possible activity sites were analysed and mutation amino acids sites to construct the mutant library were defined. 3. Constructing and evaluating of yeast display mutant library on DAF scaffold. The mutation was introduced into DAF by overlap extension PCR and megaprimer PCR. Yeast display peptides library on DAF protein scaffold were constructed. The efficiency of the protocol and the randomness of the library were determined. It was found that the constructed library were complete, the randomness was good and could be used for screening . This was the first report that yeast surface display librar...