Construction, Expression And Biological Activity Study Of Long Acting Erythropoietin

Erythropoietin (Epo) is a 34-kd glycoprotein produced mainly by kidney paratubular 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 I. Construction and identification of the recombinant human erythropoietin dimer eukaryotic expression plasmid 1. Preparation of the EPO gene The EPO mRNA was obtained from the human fetal liver and the EPO cDNA(579bp) was obtained by RT-PCR. 2. Clone or subclone recombinant EPO gene 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. 3.Construction of the recombinant plasmid pBT-2s and pBT-3c 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. 4. Construction of the recombinant plasmid pBT-2se and pBT-3ce 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 protein 1.Transient transfection of the COS-7 cell The 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. 2. Stable Transfection and selection of MTX concentration and expression promoters. 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. 3.Purification and identification of the rhEPO dimer fusion protein (1) Purification of the rhEPO dimer fusion protein purification process : Harvest cell medium →concentration and dialysis →DEAE Sepharose ion-exchanger →C4 reverse phase HPLC (2) Identification of the rhEPO dimer fusion protein Purified dimer EPO protein was shown a MW of 70KD by SDS-PAGE method. The protein was also highly reacted with specific α-EPO antibody. The purity of purified dimer EPO protein is up to 95%.Part 3. Detection of the in vivo and in vitro biological activities of the rhEPO dimer fusion protein 1.Detection of in vitro biological activity The in vitro biological activity of the rhEPO dimer fusion protein and the rhEPO monomer protein were compared by ELISA and the Clonogenic assays. The specific activity of the rhEPO dimer and monomer were 11.05 IU/Pmol and 5.65 IU/Pmol respectively. And the clonogenic number were 83±0.47,26±0.34 respectively. It indicated that the vitro biological activity of the rhEPO dimer fusion protein were far more increasing than the monomer protein. 2. Detection of in vivo biological activity In vivo biological activity of the rhEPO dimer fusion protein and the rhEPO monomer protein were compared by the erythrocyte sedimentation rate(ESR)assays.The ESR value of the mouse injected rhEPO dimer fusion protein increased 4%, and the monomer increased only 1.5%. It indicated that the vivo biological activity of the rhEPO dimer fusion protein were 1-3 times better than the monomer protein. Part 4. The primary pharmacokineties of the rhEPO dimer fusion protein Erythropoietin dimers exhibit a markedly prolonged in vivo half-life. We injected two groups of rats intravenously either with monomeric Epo or with the dimeric form purified protein. At specified times, blood samples were collected, and the Epo biological activity in the serum was determined by ELISA assay. The serum Epo levels of animals injected with monomer decreased rapidly with an approximate half-life of 0.8h. In marked contrast, the serum Epo levels of animals injected with dimer were decreased only moderately after 1.2 h. In the study, we report the design and characterization of a recombinant fusion protein made of 2 human Epo molecules linked by a peptide linker of 14 amino acids. We show that dimer has enhanced erythropoietic activity, both in vitro on primary human erythroid progenitors and in vivo in normal mouse compared with monomer. In this study, we constructed recombinant human erythropoietin dimer eukaryotic expression vector .The best plasmid pBT-2s was transfected to CHO-dhfr-cell and the expressed cell line CHO-BTE was selected by increasing the MTX. The in vivo and in vitro biological activities of the purified long acting recombinant human erythropoietin dimer fusion protein is far more than the recombinant human erythropoietin monomer protein.It can probably further expands its clinical applications.If the experimental...