Rashid Naira, Thapliyal Charu, Chaudhuri Chattopadhyay Pratima
Amity Institute of Biotechnology, Molecular Biophysics Lab, Amity University, Sector 125, Noida, Uttar Pradesh, 201313, India.
Kusuma School of Biological Sciences, Indian Institute of Technology, Haus Khas, New Delhi, 110016, India.
Int J Biol Macromol. 2017 Oct;103:1044-1053. doi: 10.1016/j.ijbiomac.2017.05.143. Epub 2017 May 25.
The process of recombinant protein production in E. coli system is often hampered by the formation of insoluble aggregates. Human Dihydrofolate reductase (hDHFR), an enzyme involved in the synthesis of purine, thymidilate and several other amino acids like glycine, methionine and serine is highly aggregation prone. It catalyzes the reduction of dihydrofolate (HF) in order to regenerate tetrahydrofolate (HF) utilizing NADPH as a cofactor. We have attempted to ameliorate the production of soluble and functional protein by growing and inducing the cells under osmotic stress condition, in the presence of various osmolytes like glycerol, sorbitol, TMAO, proline and glycine at 37°C. The expression and yield of functional hDHFR protein were highly enhanced in the presence of these osmolytes. The specific activity of the purified recombinant hDHFR protein has also been increased to a cogent level in the presence of osmolytes. We also observed that protein expressed in presence of the osmolytes was stable in the denaturing conditions as compared to the protein expressed in absence of an osmolyte. We also observed using the intrinsic fluorescence spectroscopy that the osmolytes didn't interfere with the structure of the protein and in denaturing conditions the protein expressed in presence of osmolytes had more stability. Our study is consequential in increasing the production of functional and soluble protein in the cell extract and will also be appropriate to find a therapeutic agent against many neurodegenerative diseases.
在大肠杆菌系统中生产重组蛋白的过程常常受到不溶性聚集体形成的阻碍。人二氢叶酸还原酶(hDHFR)是一种参与嘌呤、胸苷酸以及甘氨酸、蛋氨酸和丝氨酸等其他几种氨基酸合成的酶,极易形成聚集体。它催化二氢叶酸(HF)的还原,以便利用NADPH作为辅因子再生四氢叶酸(HF)。我们试图通过在37°C下,在甘油、山梨醇、三甲胺氧化物、脯氨酸和甘氨酸等各种渗透剂存在的情况下,在渗透胁迫条件下培养和诱导细胞,来改善可溶性和功能性蛋白的生产。在这些渗透剂存在的情况下,功能性hDHFR蛋白的表达和产量得到了显著提高。在渗透剂存在的情况下,纯化的重组hDHFR蛋白的比活性也提高到了一个可观的水平。我们还观察到,与在没有渗透剂的情况下表达的蛋白相比,在渗透剂存在的情况下表达的蛋白在变性条件下是稳定的。我们还使用内源荧光光谱法观察到,渗透剂不会干扰蛋白的结构,并且在变性条件下,在渗透剂存在的情况下表达的蛋白具有更高的稳定性。我们的研究对于提高细胞提取物中功能性和可溶性蛋白的产量具有重要意义,也将有助于找到一种针对许多神经退行性疾病的治疗药物。