Production and Purification of Human RecombinantGM-CSF in E. Coli

Objective: Granulocyte-macrophage colony-s timulating factor(GM-CSF) or CSF۲ is an important hematopoietic growth factorand immune modulator. GM-CSF is produced in responseto immune or inflammatory s timuli by activated cells of thehematopoietic sys tem such as T cells, B cells, macrophages,and mas t cells. Human GM-CSF (hGM-CSF) has ۱۲۷ residuesderived from a precursor containing a signal peptide. In the presents tudy, the co-expression of thioredoxin fused with hGMCSFwas accomplished to improve the production of the solubleand active form of hGM-CSF in SHuffle T۷ and BL۲۱(DE۳) E.coli s trains as the hos t cell with enhanced capacity to correctlyfold the protein.Materials and Methods: The coding sequence of hGM-CSFwas amplified by polymerase chain reaction (PCR) and finallysubcloned into the pET-Duet۱ vector. Thioredoxin (trxA) andHis-tag sequences were added at the N-terminal of the hGMCSFby designing the specific primers. After confirming the accuracyof the expression vector, the plasmid was transformedinto SHuffle and BL۲۱(DE۳) E. coli. Transformed bacteriawere cultured in LB broth containing ampicillin. Expression ofthe soluble hGM-CSF protein in LB medium was achieved byIPTG induction when the OD۶۰۰ was approximately ۰.۵. Atlas t, the recombinant protein was purified with Ni-affinity agaroseand analyzed by the SDS-PAGE method.Results: The accuracy of cons tructed vector (pET.Duet/trxA/His-tag/hGM-CSF) was confirmed and after its transformation,the optimal condition for the production of soluble recombinanthGM-CSF was obtained using ۱ mM IPTG at ۳۰ °C for ۴ hours.Eventually, the recombinant protein was successfully isolatedwith Ni-affinity resin. The efficient purification of hGM-CSFwith Ni-affinity agarose was confirmed by ۱۵% SDS-PAGE inwhich a protein band with the size of ۱۴.۴۷ kDa was observed.Conclusion: In the present s tudy, efficient Shuffle E. coli expressingsoluble recombinant hGM-CSF were obtained. Theresults showed that the recombinant protein efficiently producedthe soluble hGM-CSF. In comparison to previous s tudiesco-expression of TrxA reduced the induction time in Shuffle E.coli, resulting in the production of more soluble recombinantprotein.

کلیدواژه ها:

Eshersi Coli (E. coli) ، Human Granulocyte-MacrophageColony-S timulating Factor (hGM-CSF) ، Thioredoxin(TrxA)