1. 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
- Author
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Sandeep Vemula, Sushma Vemula, Akshay Dedaniya, Rajesh Kumar Kante, and Srinivasa Reddy Ronda
- Subjects
0301 basic medicine ,medicine.disease_cause ,Biochemistry ,Protein Structure, Secondary ,Inclusion bodies ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Sodium pyruvate ,law ,Granulocyte Colony-Stimulating Factor ,Spectroscopy, Fourier Transform Infrared ,Escherichia coli ,medicine ,Humans ,Thermal stability ,Fourier transform infrared spectroscopy ,Protein secondary structure ,Temperature ,General Medicine ,Recombinant Proteins ,Culture Media ,Granulocyte colony-stimulating factor ,030104 developmental biology ,chemistry ,Recombinant DNA ,Electrophoresis, Polyacrylamide Gel ,Biotechnology - 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.
- Published
- 2016
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