Investigation Of Annexin A1 As A Multifunctional Lable

In the affinity purification of recombinant proteins,the modified and optimized affinity tag is fused with the target protein for expression.Through simple and fast affinity chromatography,higher purity fusion protein was obtained directly,and the purity fusion protein has the advantages of high binding specificity,mild purification conditions and wide applicability.At present,the main problem of affinity chromatography is high cost and the operation process is relatively cumbersome.The purpose of this study is whether the calcium ion dependent property of Annexin A1 can be used as an affinity tag to obtain purified recombinant proteins efficiently and cheaply by non-affinity chromatography.Then we can study the structure and function of proteins and use them to make protein drugs.For this purpose,this paper conducted relevant experimental:1.Construction of expression vectors: prokaryotic expression vectors were constructed to fuse five proteins(Em GFP,ALAD,y HAD,m SR,Sir B)at the C-terminal,and all five fusion proteins fused the histidine tags at the N-terminal.In order to remove the fusion proteins later,These five proteins all contained the Tev protease recognition sequence ENLYFQG,and the vector of Tev protease was also constructed.In order to increase the solubility of the fusion protein,MBP was fused at the N-terminal and green fluorescent protein was fused at the C-terminal.The vector suitable for expression in pichia pastoris of eukaryotes was also constructed and the green fluorescent protein was fused at the C-terminal.2.Nickel column affinity chromatography: According to the strong attraction of the side chain of histidine residue and solid nickel,it can be used for IMAC.The recombinant protein was isolated and purified,and the purified target protein was obtained.But the operation is relatively complex.3.Heparin agarose affinity chromatography: Annexin A1 and heparin binding properties were used for affinity chromatography.The target protein obtained by heparin affinity chromatography was of high purity and yield,The protein yield by heparin affinity chromatography was 24.5%,but its cost was high.4.Rapid affinity precipitation of calcium ions: according to the properties of the Annexin A1,calcium ions were used to conduct affinity precipitation of the recombinant protein,then the target protein was isolated using the metal ion chelating agent EDTA.The resulting target protein was of high purity,simple operation and low cost.The yield of recombinant protein is about 20%.5.Rapid affinity precipitation of yeast expressing target protein: In this paper,p PICZαA,which can transport the protein of interest to the outside of the cell,was selected as the expression vector.Hope to get more target protein in the medium for further affinity precipitation,However,no protein expression was detected in the yeast medium.The target protein was detected after yeast solution was broken,Analysis by affinity precipitation did not achieve the desired results.The yield of protein affinity precipitated in yeast was lower,only 5.2%.To sum up,Annexin A1 can be used for fast affinity precipitation of calcium ions to obtain high purity recombinant proteins.Compared with nickel column affinity chromatography and heparin agarosaccharide chromatography,it is easy to operate,low cost and easier to obtain purified target proteins.Moreover,the protease obtained by rapid affinity precipitation of calcium ions still has high biological activity.However,the target protein was not secreted into the culture medium in the yeast expression,which was not consistent with the expected results in the yeast system.In summary,Annexin A1 can be used as an affinity tag to isolate and purify target proteins in Escherichia coli.