In E. coli, the formation of inclusion bodies (IBs) is commonly seen upon overexpression of recombinant proteins. The recombinant protein inside IBs is usually misfolded and a series of denaturation/renaturation steps are necessary for isolation of biologically active protein. In recent study, nonclassical inclusion bodies (nIBs) - a new subtype of IBs - containing a high percentage of correctly folded protein can be extracted under non-denaturing conditions. The absence of denaturation/renaturation step allows to develop more efficient process with lower costs and environment-friendly technologies. In the studies previously published, the authors have developed a simple, efficient and applicable process for extraction of biologically active hG-CSF protein without renaturation procedure required. The expression of rhG-CSF in E. coli BL21 (DE3)/pET-gcsf was induced by 0.5mM IPTG at low temperature. The cultural medium was supplemented with isopropanol, stress causative agent. The nIBs were isolated and resuspended with 0.2 percent (w/v) N-lauroylsarcosine. The expression and solubilization of nIBs were confirmed by SDS-PAGE. Characterization of rhG-CSF by reverse-phase HPLC showed similar result to standard. At one-liter scale-up, 33.04 mg ofbioactive hG-CSF protein/per liter of cell culture were achieved. The solubilized rhG-CSF was successfully purified by IMAC (Immybilized metal ion affinity chromatography) with the yield of 91.36 percent. The biological activity of rhG-CSF was ascertained via proliferative assay using the murine myeloblastic M-NFS60 cell line accomplishing 9.24 x 10 exponent 7 IU/mg specific bioactivity with 91.7 percent purity.
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