Segmental Expression And Purification Of Mouse PR Domain Containing Protein 16(PRDM16)in Escherichia Coli

Mouse PR domain protein 16(PRD1-BF1 and RIZ1 homeodomain protein 16,PRDM16)is an important transcription factor in the differentiation of brown adipocytes and is a member of PRDM family with PRDM2,PRDM3,PRDM6,PRDM8,and PRDM13.The mouse PRDM16 gene is located on the long arm E2 of chromosome 4,and mainly contains one N-terminal PR domain and two C2H2-type zinc finger domains.The mature PRDM16 encodes 1176 amino acids with molecular weight about 150-170 KDa.It is mainly expressed in brown adiposite,testes,heart,kidneys and lungs.The main biological functions of the PRDM16 are the effects of energy homeostasis,glucose and lipid metabolism,and body weight regulation.It is also the "switch" for the conversion of white fat and brown fat.In addition,it plays an important role in regulating oxidative stress,the establishment of hematopoietic stem cells,and the development of organs such as the heart.In this study,the long PRDM16 cNDA was divided into 6 parts,then the PRDM16 fragment was amplified with PCR and inserted into the pMalC2 X vector behind Lactose operon respectively to obtain pMalC2X-PRDM16-(1/2/3/4/5/6)-Flag recombinant vectors.Next the His tag and the TEV tag were inserted containing the GSG flexible chain separately into the upstream and downstream of the MBP to get His-pMalC2X-TEV-PRDM16(1/2/3/4/5/6)-Flag vector.After this plasmid vector was transformed into E.coli expression strain BL21(DE)3,the high expression strain was screened out.PRDM16(6)target protein was induced by IPTG and was expanded culture at the explored optimal induction conditions.After lysis,cell supernatant passed through the Ni affinity column and DEAE DEAE anion exchange column,the purity of the target protein can reach 87.1%.The results of western blotting showed that the size of the protein was in line with the forecast.,which further confirmed the correct expression of the target protein.Then PDM16 fusion polypeptide with GST tag was expressed and purified in the same way.The expression and purification of these fusion proteins could lay a foundation for the preparation of PRDM16 specific antibody.