Study In Construction, Expression And Purification Of Dimer Fusion Erythropoietin

Erythropoietin is a 34-kd glycoprotein produced mainly by kidneyparatubular cells in response to reduced oxygen delivery. Epo stimulates erythroid progenitor cell proliferation, differentiation and maturation. It also inhibits cell apoptosis, which results in increased erythrocyte formation. The recombinant human erythropoietin (rhEpo) is widely used to compensate for the reduced production of endogenous Epo in renal failure and to correct the associated anemia. Administration of rhEpo alleviates the necessity for blood transfusion and greatly improves the quality of life for patients. Clinical studies have shown that rhEpo can also be effective in the treatment of other chronic anemias. Injections of high doses of rhEpo have been shown to increase blood hemoglobin levels and occasionally to alleviate the need for transfusion.Various strategies have been pursued to increase the yield of Epo production and to enhance its intrinsic activity. For instance, codon optimization of the Epo DNA sequence resulted in greater protein production. In another strategy, the purification of rhEpo beared an elevated number (up to 14) of sialic acid residues . Epo mimetic peptides are small and relatively easy to produce in large quantity, but their activity is still very poor compared with that of native Epo. It was found that the association of two Epo molecules obtained by either chemical cross-linking or recombinant DNA-mediated fusion of coding regions resulted in a more stable protein than the native monomer with an increased in vivo life span.In this study, we report the design and characterization of a recombinant fusion protein made of two human Epo molecules linked by a peptide linker of 14 amino acids. We show that this dimer has enhanced erythropoietic activity, both in vitro on primary human erythroid progenitors and in vivo in normal mouse compared with its monomer counterpart. In this study, we construct three recombinant human erythropoietin dimer eukaryotic expression vectors with different promotor, pBT-1c , pBT-2s ,pBT-3c and two recombinant human erythropoietin monomer eukaryotic expression vector pBT-2se,pBT-3ce. Plasmid pBT-2s was transfected to CHO-dhfr- cell and the expressed cell line CHO-BTE was selected by increasing the methotrexate (MTX) . The expression level of EPO dimer was up to 33.3 g /106cell?d. The in vivo ,in vitro biological activities and the half life (20h) of the purified recombinant human erythropoietin dimer fusion protein is far more exceeded than the recombinant human erythropoietin monomer protein.The project includes three parts as following.Part 1. Construction and identification of the recombinant human erythropoietin dimer eukaryotic expression plasmidThe EPO mRNA was obtained from the human fetal liver and the EPO cDNA(579bp) was obtained by RT-PCR.Two different EPO gene fragments with parts of linker were obtained from the EPO cDNA by using the primers P3/P4 and P5/P6 respectively. The fragments were introduced at the NheI and HindⅢsites and plasmid pGEMTE was constructed by ligation of the linker fragment encoding 14 amino acid residues and the EPO gene into the T vector.The pBT-1c was obtained by removing the internal EPO dimer fragment (1119bp)from pGEMTE. The sequences were verified by sequencing and were found to be in agreement with the expected sequence.The EPO dimer gene was introduced into the HindⅢand XhoI site by PCR from the pGEMTE plasmid.The plasmid pBTsv and pBTcmv were digested by HindⅢand XhoI. Then the plasmid pBT-2s and pBT-3c were obstained by ligation of the two fragments. The sequences were verified by sequencing and were found to be in agreement with the expected sequence.The EPO gene fragment was introduced into the HindⅢand XhoI site by PCR from the EPO cDNA. The plasmid pBTsv and pBTcmv were digested by HindⅢand XhoI. Then the plasmid pBT-2se and pBT-3ce were obstained by ligation of the two fragments. The plasmid were verified by sequencing and were found to be in agreement with the expected sequence.Part 2. Tansfection, expression and purification of the rhEPO dimmer fusion proteinThe culture supernatant were collected after the transfection of COS-7 cell by the three different rhEPO dimer expression vectors pBT-1c , pBT-2s ,pBT-3c in the 24h and 72h. And the transient transfection expression level were detected by the Dot Blotting and ELISA assay, the level were 33 ng/ mL, 95.8 ng/ mL,60 ng/ mL respectively. Both of the results indicated that the transient transfection expression level of the plasmid pBT-2s were most.The rhEPO dimer expressed plasmid pBT-1c, pBT-2s, pBT-3c and the rhEPO monomer expressed vector pBT-2se, pBT-3ce were transfected into the CHO-dhfr- cell respectively, specially,the plasmid pBT-1c and pSV2 were Co-transfected into the CHO-dhfr- cell.The mixed expression clones were obtained in the selective medium with MTX. Then the number of the clones and the modality of the cells were compared between the cells transfected by EPO dimer vector and by monomer vector.Then mixed the clones and continued to co-amplify the foreign gene in the selective medium with gradingly increasing concentrations of MTX to 100nM and detected by ELISA. Finaly,we selected the mixed clones transfected by the plasmid pBT-2s to keep the stepwise increasing MTX concentrations,by all of the results After two round of cloning of the expression cells , high-level of stable expression cell line was obtained with expression level of 33.3 g /106cell?d.Part 3.Purification and identification of the rhEPO dimer fusion protein:purification process : Harvest cell medium→concentration and dialysis →DEAE Sepharose ion-exchanger→C4 reverse phase HPLCPurified dimer EPO protein was shown a MW of 70KD by SDS-PAGE method. The protein was also highly reacted with specificα-EPO antibody. The purity of purified dimer EPO protein is up to 95%.Research characteristics:1.rhEPO dimer fusion protein is a new kinds of erythropoietin,it can cure renal failure and all kinds of anemia.2.The biological activity and the half life of the long acting rhEPO dimer fusion protein were far more increasing than the monomer protein.Innovation features:1.It was found that the association of Epo molecules obtained by chemical cross-linking can manufacture complex products of admixture of huge molecular.so we obtained the rhEPO dimer fusion protein by gene engineering in this study.2.In this study , we constructed recombinant human erythropoietin dimer eukaryotic expression vector and selected the best vector .3.We have found the best transfect way and the best MTX stepwise increasing method, and got the high expressed cell line.4.Our purification process is rapid and efficient with high recovery.5.The biological activity of the rhEPO dimer fusion protein were far more increasing than the monomer protein.