title: Characterization of recombinant human granulocyte colony-stimulating factor expression by FT-IR spectroscopy: Studies on thermal induction and media formulation on the stability of the protein secondary structure.
abstract: The Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline + sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46 mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline + sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.
authors.affiliation: a Centre for Bioprocess Technology, Department of Biotechnology , K. L. E. F. University , Guntur , Andhra Pradesh , India.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Vemula
authors.affiliation: b Department of Pharmacology, Kakatiya University , Warangal , Andhra Pradesh , India.
authors.forename: Sushma
authors.initials: S
authors.lastname: Vemula
authors.affiliation: a Centre for Bioprocess Technology, Department of Biotechnology , K. L. E. F. University , Guntur , Andhra Pradesh , India.
authors.forename: Akshay
authors.initials: A
authors.lastname: Dedaniya
authors.affiliation: a Centre for Bioprocess Technology, Department of Biotechnology , K. L. E. F. University , Guntur , Andhra Pradesh , India.
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Kante
authors.affiliation: a Centre for Bioprocess Technology, Department of Biotechnology , K. L. E. F. University , Guntur , Andhra Pradesh , India.
authors.forename: Srinivasa Reddy
authors.initials: SR
authors.lastname: Ronda
mesh_terms: D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013696:Temperature
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor
keywords: FT-IR; inclusion bodies; media formulation; temperature shifting strategy; thermal stability