Site-Directed Mutagenesis in Human Granulocyte-colony Stimulating Factor, Cloning and Expression in Escherichia coli

Abstract

Human granulocyte colony stimulating factor (hG-CSF) induces proliferation and differentiation of granulocyte progenitor cells. This glycoprotein is currently being used for treatment of neutropenia, in patients who have undergone bone marrow transplantation. So far, different researchers have tried to enhance hG-CSF biological activity and stability. In this study, Polymerase Chain Reaction (PCR) based site-directed mutagenesis was performed on hG-CSF cDNA. The final amplified DNA fragment was cloned into the pBluescript sk(-) plasmid and after verification of the desired mutations by sequencing, it was subcloned into the pET-21a(+) vector and expressed in Escherichia coli BL21. The mutant G-CSF product was analyzed by SDS-PAGE and Western-blot analyses. The results show that the recombinant mutant G-CSF has been cloned and expressed successfully in prokaryotic system. This research aimed to produce a new recombinant hG-CSF expected to show enhanced biological characteristics in contrast to those of the native hG-CSF. The analysis of its function and biological characteristics remain to be examined.