Prokaryotic Expression And Purification Of Recombinant CARD9-MBP Fusion Protein

Caspase recruitment domain-containing protein 9(CARD9)is a nonredundant adapter protein involed in signal transduction.It located in chromosome 9q34.3 and the total length of cDNA is 2108 bp.CARD9 encodes a 536-amino acid protein with a molecular mass of 62.3 kDa,furthermore,it can form CBM complex with Bcl 10 and MALT 1,and connect pattern recognition receptors.CARD9 is a key signaling molecule for innate immune responses,which plays an important role in recognizing and resisting the fungi,bacteria and virus infection.Encoded byE.coli malE gene,MBP is a member of the bacterium maltose transporter system and mainly involved in the uptake and catabolism of maltose in E.coli.MBP is composed of 371 amino acid residues,whose molecular mass is about 42 kDa.MBP-tag is mainly used to promote the solubility of protein,which has the advantages of high solubility,high efficiency and easy purification.It can increase the solubility of overexpressed fusion proteins in bacteria,especially eukaryotic proteins.Besides,MBP-tag can increase the stability of the target protein.After separation from the MBP-tag,the target protein can easily recover its natural conformation.Since CARD9 plays an important role in innate immune response,it is particularly important to analyze its protein structure and clarify its function.So far,the research on CARD9 has focused on the immune and animal experiments in vivo,while the report about its protein structure has not been published.Therefore,this project tries to establish the experimental basis for further research by constrructing a prokaryotic expression vector of CARD9-MBP fusion protein,and to express and purify it.ObjectiveTo construct a prokaryotic expression vector of CARD9-MBP fusion protein,and the fusion protein was expressed and purified.MethodsThe coding sequence of CARD9 and MBP genes were amplified by PCR and the recombinant plasmid was correctly inserted into the pET-3 0a(+)vector by enzymes restriction and ligation.The recombinant plasmid was transformed into E.coli DH5a competent cells and proceeded PCR identification,restriction analysis and gene sequencing.The correct recombinant plasmid was transformed into E.coli BL21(DE3)competent cells.The target protein was induced to express by IPTG under different conditions.Relative molecular weight of the target protein was detected by SDS-PAGE electrophoresis.The CARD9-MBP fusion protein was purified by MBP maltose chromatography column and gel filtration chromatography column,and MBP-tag attempted to be removed by HRV3C enzyme overnight.ResultsThe CARD9-MBP fusion protein was successfully constructed and confirmed by PCR and restriction analysis.The result of gene sequencing was consistent with the target sequence.The SDS-PAGE electrophoresis showed that the target protein with molecular mass(MR)about 105 000 was successfully induced to express in E.coli BL21(DE3).A quite pure CARD9-MBP fusion protein was obtained by purification of MBP maltose chromatography column.After removal of MBP-tag by HRV3C enzyme overnight,CARD9 protein mainly exists in sediment.In the later stage,the initial purified protein was mixed,concentrated,and purified by gel filtration chromatography column to obtain further pure CARD9-MBP fusion protein.ConclusionThe prokaryotic expression vector of CARD9-MBP fusion protein was successfully constructed and a large number of soluble protein expressed.The purified target protein can be obtained by purification with MBP maltose chromatography column and gel filtration chromatography column.