Archive \ Volume.10 2019 Issue 4
Cloning and expression of recombinant human granulocyte-macrophage colony stimulating factor (rhGMCSF) in Escherichia coli Leila mehran-nia , Nosratollah Zarghami , Masoumeh Mehrpouya , Majid Shahbazi
Introduction: Human GM-CSF is glycosylated and consists of 127 amino acids with an approximately MW of 14/5 KDa. Because of its effects on separating hemopoietic cell population, it is clinically used to improve haematopoiesis in oncologic disorders. Some clinical applications of GM-CSF include the treatment of myelodysplasia syndromes, myelosuppression induced by chemotherapy or a marrow transplantation, aplastic anaemia and AIDs, and hence used as a vaccine adjuvant. After optimizing and synthesizing human GMCSF genes, it cloned at pUC18 vector and E.coli (DH5α) host. After enzymatic digestion, the gene was inserted into expression vector under the control of T7 promoter and was subcloned into E.coli Bl21 (DE3). Cells were grown in LB broth medium, induced by IPTG, the expressed protein in the form of inclusion body extracted using DDT regenerative material. Considering the huge therapeutic potential of GM-CSF, the development of strategies for production of this protein at low costs is desirable so that the drug is made available to a vast population. Therefore, we used Escherichia coli to produce rhGMCSF. Colony PCR analysis showed correct translocation of gene in correct format in expression vector which showed the existence of 596bp GM-CSF gene. Western blot and SDS-PAGE confirmed GM-CSF protein expression.