Production and Simple Purification of Recombinant Human Granulocyte Colony-Stimulating Factor using the Inteintag in Escherichia Coli

Background: Granulocyte colony-stimulating factor (G-CSF) is a cytokine which have many functions such as stimulating of hematopoiesis, proliferation and differentiation of granulocyte progenitor cells and production of bone marrow neutrophilic granulocyte colonies. Nowadays, the usage of human recombinant G-CSF (rh G-CSF) is for curing of chemotherapy- radiotherapy-induced neutropenia, and also in patients with bone marrow transplantation. The aim of this study was to produce a soluble type of G-CSF in E.coli and purification in new way. In this study, we achieved active G-CSF using the intein fusion system. Therefore, two copies of optimized Intein - GCSF genewas cloned into the pET22b vector to generate pET22b-G-CSF-intein2 (C-terminal fusion vector) and pET22b intein1-G-CSF (N-terminal fusion vector). The hG-CSF sequence with two different intein sequence from pTWIN1 vector are synthesized and then inserted into a pET22 expression vector under the control of T7 promoter and cloned in E. coli strain BL21 (DE3).The fused proteins formed misfolded proteins as inclusion bodies (IBs). The IBs were solved with urea solution and afterward proteins were saved for size-exclusion chromatography. Then , CBD-intein–GCSF was loaded onto chitin beads column equilibrated with 10mM Tris buffer, 500mM NaCl, pH 8.5, and was eluted from the column by incubation at 25°C under pH 6.5 for 16h based on intein C-terminal self-cleavage. SDS-Page, western blot, dot blot, size exclusion chromatography and in vivo assay of protein demonstrated that the bioactivity of recombinant GCSF was compared with standard GCSF. Results: After culturing and induction of recombinant E. coli with IPTG, we obtained a good expression of the hG-CSF, as determined by SDS-PAGE and confirmed by dot blotting and western blotting. Approximately 50% of the expressed G-CSF was soluble after the IPTG induction and temperature reduction. Up to 1.2 mg of G-CSF was achieved from a 1 L culture, and nearly75% of the GCSF-Intein2 was cleared by pH shift. Intein1-GCSF was not cleaved due to the inhibition of cleavage by the N-terminal amino acid of GCSF. The biological activity assay, in vivo, showed a higher biological effect than standard reference hG-CSF. Conclusion: The immunological and biological analyses showed that this protocol can be useful to develop bioproducts. In conclusion, the combination of different methods presented here permitted an simple and cost-effective protocol for rhG-CSF production.