Cloning and expression of Fh8 tag-fused G-CSF recombinant protein

Recombinant protein drugs (rPDs) increased much attention in recent years. Optimizing and uniformity of the production process is of great interest.  Granulocyte colony-stimulating factor (G-CSF) is a protein used to treatment of neutropenia caused by chemotherapy drugs. The rPD production's low efficiency, low solubility in Escherichia coli (E. coli) as an expression host, and purification are difficulties of these systems. Consequently, this study aimed to clone and express the Fh8 tag-fused G-CSF recombinant protein to investigate the effect of this tag on the solubility of G-CSF. The Fh8 gene and the G-CSF were cloned inside pET28 and pET23 plasmids in previous studies, respectively. Following isolating the G-CSF gene by PCR and transferring it to T Vector, plasmids pET28 and T Vector were cut using BamHI and NotI enzymes. G-CSF gene was inserted into pET28 and next to FH8, with the T4 ligase enzyme assist. The fused gene's presence in the recombinant vector was investigated using the PCR technique; expression of the recombinant proteins was also evaluated using SDS-PAGE and Western blotting. PCR and fragment analysis results on gel electrophoresis indicated that the expression vector was correctly cloned in E. coli. Also, analysis of protein expression using SDS-PAGE, and Western blotting methods indicated that this host can express the desired gene construct. Accordingly initial evaluations, the use of the FH8 tag increases GCSF solubility. E. coli is a proper host for the expression of GCSF-FH8 fused protein. Further examination is needed to confirm FH8's potential to increase GCSF protein solubility.