A Biochromatography Based On M1.3 Origami Assembled Peroxisome Proliferators-activated Receptor ?: Construction And Application

Traditional Chinese Medicines are important drug resources with high development values in our country.It will be of great importance in screening active ingredients acting on PPAR? from traditional Chinese medicines.However,the traditional drug screening models are costly and complicate,we need to construct an efficient,rapid,cost-effective drug screening model.In recent years,researchers have made rapid progress in biological chromatography.Silica is a desired support to immobilize receptors.However the lack of biocompatibility remains an obstacle to its widespread use.M1.3 origami,a small origami which has the advantage of being cost-effective and biocompatible,it can recover the disadvantages of silica.In this study,a biochromatography based on M1.3 origami assembled PPAR? was constructed.Firstly,we mainly investigated the effects of additional Mg2+ and JM109 on the yield of M13mp18 ssDNA and optimized the extraction process of M13 ssDNA.The result showed that the yield of M13 ssDNA was increased with the presence of additional JM109 cells,Mg2+.Short double stranded regions-cleavage sites were constructed by annealing two oligonucleotide strands with M13 ssDNA.Restriction endonucleases EcoRI and BglII were used to prepare the M1.3ssDNA with 704 nucleotides.The recovered M1.3ssDNA fragment was mixed with the modified and unmodified staple strands in folding buffer,M1.3 origami was obtained after the annealing process.The results of agarose gel electrophoresis and transmission electron microscopy showed that M1.3 origami was assembled successfully.The recombinant plasmid pET28a-PPAR?-LBD was transformed into the E.coli BL21(DE3)cells.IPTG was added to induce protein expression.The hPPAR?-LBD recombinant protein with His tag was extracted and purified by Ni-NTA-Sefinose.The results of SDS-PAGE showed that hPPAR?-LBD recombinant protein was successfully extracted.The receptor was modified with the oligonucleotide strand which was complementary to the M1.3 origami free base.The receptor protein was arranged on the surface of M1.3 origami by the principle of complementary basepairing;M1.3 origami-PPAR? was bonded to the surface of thiolated silica with the aid of SMCC.The silica gel surrounding by M1.3 origami-PPAR? was packed into the column(50 mm × 2.1 mm I.D.)by a wet packing method to obtain M1.3origami-PPAR? biochromatography column.Secondly,the affinity,specificity and stability of M1.3origami-PPAR?biochromatography column were studied.The synthetic ligand rosiglitazone and natural ligand curcumin were used as positive drugs.The retention behaviors of these two positive drugs were studied to evaluate the affinity of M1.3origami-PPAR?biochromatography column compared with the negative column.Phenylbutazone was used as the negative drug to show the specificity of the model.Curcumin was used to evaluate the stability of M1.3 origami-PPAR? column within one month.The results demonstrated that rosiglitazone and curcumin retained well on the M1.3origami-PPAR? column,which indicated that the biochromatography column had the affinity to the PPAR? ligand.The retention behavior of the mixed solutions of phenylbutazone and rosiglitazone showed that the biochromatography column had the ability to screen the active gradients acting on PPAR? from mixed solutions.The retention behavior of curcumin on the biochromatography column within one month indicated that the stability of the column reduced gradually.Lastly,the biochromatography column was used to screen active ingredients from Ginsenosides,gardenia and rhubarb.The results showed that these three medicines could retain on the column.The retention fraction of Ginsenosides was analyzed by HPLC-MS.And the retention fraction of gardenia was was analyzed by HPLC.The results indicated that Ginsenoside Re and geniposide were the active composations.The active ingredients of rhubarb have not been identified yet.In this study,the M1.3 origami-PPAR? biochromatography column has the advantage of being efficient,rapid and cost-effctive.It is possible to screen the active ingredients from complex components directly without the separation of the drugs,thereby greatly save the time and cost of drug screening.