title: Improved Production and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor from E. coli under Optimized Downstream Processes.
abstract: This work reports the upstream and downstream process of recombinant human granulocyte colony stimulating factor (rhG-CSF) expressed in Escherichia coli BL21 (DE3)pLysS. The fed batch mode was selected for the maximum output of biomass (6.4g/L) and purified rhG-CSF (136mg/L) under suitable physicochemical environment. The downstream processing steps viz., recovery, solubilization, refolding and concentration were optimized in this study. The maximum rhG-CSF inclusion bodies recovery yield (97%) was accomplished with frequent homogenization and sonication procedure. An efficient solubilization (96%) of rhG-CSF inclusion bodies were observed with 8M urea at pH 9.5. Refolding efficiency studies showed maximum refolding ⩾86% and ⩾84% at 20°C and pH 9 respectively. The renatured protein solution was concentrated, clarified and partially purified (⩾95%) by the cross flow filtration technique. The concentrated protein was further purified by a single step size exclusion chromatography with ⩾98% purity. The characterization of purified rhG-CSF molecular mass as evidenced by SDS-PAGE, western blot and LC/MS analysis was shown to be 18.8kDa. The secondary structure of rhG-CSF was evaluated by the CD spectroscopic technique based on the helical structural components. The biological activity of the purified rhG-CSF showed a similar activity of cell proliferation with the standard rhG-CSF. Overall, the results demonstrate an optimized downstream process for obtaining high yields of biologically active rhG-CSF.
authors: K L E F University, Centre for Bioprocess Technology, Department of Biotechnology, Guntur 522 502, Andhra Pradesh, India.
authors: Sandeep
authors: S
authors: Vemula
authors: K L E F University, Centre for Bioprocess Technology, Department of Biotechnology, Guntur 522 502, Andhra Pradesh, India.
authors: Rahul
authors: R
authors: Thunuguntla
authors: K L E F University, Centre for Bioprocess Technology, Department of Biotechnology, Guntur 522 502, Andhra Pradesh, India.
authors: Akshay
authors: A
authors: Dedaniya
authors: K L E F University, Centre for Bioprocess Technology, Department of Biotechnology, Guntur 522 502, Andhra Pradesh, India.
authors: Sujana
authors: S
authors: Kokkiligadda
authors: K L E F University, Centre for Bioprocess Technology, Department of Biotechnology, Guntur 522 502, Andhra Pradesh, India.
authors: Chaitanya
authors: C
authors: Palle
authors: K L E F University, Centre for Bioprocess Technology, Department of Biotechnology, Guntur 522 502, Andhra Pradesh, India. Electronic address: rsr@kluniversity.in.
authors: Srinivasa Reddy
authors: SR
authors: Ronda
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor
keywords: Characterization; Cross flow filtration; Fed-batch fermentation; Inclusion bodies; Refolding kinetics; Reoxidation