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

Sandeep R Vemula, Sushma Vemula, A. Dedaniya, Rajesh Kumar Kante, Srinivasa Reddy Ronda
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Abstract

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.
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重组人粒细胞集落刺激因子表达的FT-IR表征:热诱导及培养基配方对蛋白二级结构稳定性的影响
采用傅里叶变换红外(FT-IR)光谱方法研究了重组人G-CSF (rhG-CSF)蛋白的积累、二级结构和热稳定性,这些重组人G-CSF (rhG-CSF)蛋白在不同培养基中在温度变化策略(37°C和28°C)下生长。胆碱+丙酮酸钠(37°C)和丙酮酸钠(28°C)配方的包涵体(IB)积累量最高,分别为0.41和0.46 mg/mL。此外,通过二级结构分析研究了IBs和完整细胞中rhG-CSF的结构。热稳定性实验也解释了rhG-CSF二阶导数的结构模式。研究表明,胆碱+丙酮酸钠配方即使在82℃下也能保持蛋白质二级结构。总的来说,FT-IR光谱技术也可以用于加速大肠杆菌来源的其他重组治疗性蛋白的表征。
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