Cloning, Expression, Purification and PEGylation of Recombinant Human Granulocyte Colony-Stimulating Factor and Biology Activity in Vitro and in Vivo

Abstract: Objective: To construct the recombinant vector which can express Recombinant human granulocyte colony-stimulating factor(rhG-CSF), which was expressed in Escherichia coli in form of inclusion body was modified by monomethoxy polythylene glycol(mPEG20k-NHS) after denaturation, renaturation and purification. The biologic activity of both the unmodified and modified G-CSF in vitro and in vivo was tested. Methods: The target gene G-CSF was inserted into pJGW-2 to construct the expression vector. The recombinant plasmid was transformed into DH5α. The pJGW-2-G-CSF/ DH5αwas induced by temperature and G-CSF protein was analysed by SDS-PAGE and identified by Western blotting. In order to obtain active G-CSF protein,inclusion bodies should be denatured and then renatured.After refolding, G-CSF was purified by SP-Sepharose FF and Sephcryl S-200. The purified G-CSF was mono-modified by PEG20k, then the modified G-CSF was also purified by SP-Sepharose FF and Sephcryl S-200 to obtain the purified PEG-G-CSF. The purity of PEG-G-CSF was measured by HPLC. The biologic activity of both the unmodified and modified G-CSF in vitro and in vivo was tested. Results: Results showed that G-CSF was expressed in 19kD in DH5αand the expression level accounted by 40% of the whole protein of the bacteria. Most of the target proteins were expressed as inclusion bodies. The recombinant protein could specifically react with a rabbit monoclonal IgG antibody against human G-CSF by Western blotting. The purity of the G-CSF protein was about 95% after purification. The optimal reaction of this PEG- G-CSF formation was conditioned as of 1∶6 mass ratio of G-CSF and PEG at pH6.0, the reaction balance in 48 hours .At this condition the ratio of PEG-G-CSF was about 45%. The purity of PEG-G-CSF can be more than 95% through purification. Compared with unmodified G-CSF(1.0×108u/mg), the modified rhG-CSF(PEG-G-CSF)had a lower biologic activity(2.0×107u/mg) in vitro, but had a significantly prolonged action time in vivo with obviously elevated leucocyte in blood plasma. Conclusions: The expressed plasmid was correctly constructed. G-CSF was expressed in E.coli, and the G-CSF was refolded and purified successfully. Set up the techniques of producing PEG- G-CSF; the G-CSF can be modified by PEG to obtain PEG- G-CSF, the purity can be more than 95% after purification. The biologic activity of the modified G-CSF desended in vitro but in vivo was enhanced…
Key words: expression; inclusion bodies; refolding; modified; biological activity

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