Preparation And Functional Characterization Of Soluble Human PPARγ-LBD Recombinant Protein

Cancer is one of the most difficult of the obstinate illness for human to overcome. There are many kinds of anticancer drugs for clinical therapy, which are still to be faced with problems of toxicity and poor selectivity. Therefore, the development of highly efficient and low toxicity anticancer drugs is very important.In recent years, at home and abroad the study of anticancer drug is turning from traditional cytotoxic anticancer drugs to the anticancer drugs which are for multi-link targets of tumor signal transduction pathways. One of the hot spots is the study of PPARγsubtypes(peroxisome proliferater-activated receptorγ, PPARγ), which is the key protein in the signal transduction of lipid metabolism. In order to study on the hPPARγ-LBD(human peroxisome proliferater-activated receptorγLigand binding domain, hPPARγ-LBD) recombinant proteins. Now,it is very difficult to obtain abundant high purity recombinant protein with the biological function. Therefore, we constructed pReceiver-B01-hPPARγ- LBD expression vector with the tag his6 in the paper, prepared the soluble hPPARγ-LBD recombinant protein through expression and purification, determined the binding activity of the hPPARγ-LBD recombinant protein and its ligand drugs with the size exclusion chromatography - high performance liquid chromatography (SEC-HPLC) in order to advance the drug design and screening based on lipid metabolism target proteins hPPARγ-LBD.The main work done and conclusion definited in the topic included these parts as below:1. The hPPARγ-LBD recombinant protein was prepared. Its molecular weight is about 32 kDa by SDS-PAGE analysis,which is consistent with the theoretical molecular weight calculated by amino acid sequence.2. The expression conditions of hPPARγ-LBD recombinant protein was optimizated. Under optimal conditions of 16℃, 0.6 mmol/L IPTG and induction time 20 h, the soluble recombinant protein was expressed successfully.3. The purification conditions of hPPARγ-LBD recombinant protein was optimizated. hPPARγ-LBD recombinant protein with weight of 41 mg, purity of 95% could obtain in the LB medium per liter after purified by nickel-affinity chromatography.4. The SEC-HPLC was establishied. The binding activity of hPPARγ-LBD recombinant protein with ligand drugs in vitro was inspected. The recombinant PPARγ-LBD could bind specifically with rosiglitazone with binding rate of 65% .In summary, we prepared hPPARγ-LBD recombinant protein with biological function in the paper. And it may lay the foundation for studying on hPPARγ-LBD target, designing or screening new anticancer drugs.