Wang Qi, Liu Yongdong, Zhang Chun, Guo Fangxia, Feng Cui, Li Xiunan, Shi Hong, Su Zhiguo
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No.1 Beierjie Street, Zhongguancun, Haidian District, Beijing 100190, PR China.
Protein Expr Purif. 2017 May;133:152-159. doi: 10.1016/j.pep.2017.03.014. Epub 2017 Mar 18.
Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity.
从包涵体（IBs）中进行蛋白质复性时，在高蛋白浓度下常常会遇到回收率低的问题。在本研究中，我们证明了高静水压（HHP）能够同时实现重组人睫状神经营养因子（rhCNTF，一种潜在的神经退行性疾病治疗药物）包涵体的高复性浓度和高复性产率。采用稀释复性法，即使在存在4M尿素的情况下，在3mg/mL时回收率也仅为18%。相比之下，HHP复性即使在4mg/mL时也能有效地将回收率提高到近100%。研究发现，在稀释过程中，疏水聚集体是偏离正确途径的产物，其数量随蛋白质浓度的增加而增加。然而，HHP能够有效地减少疏水聚集体的形成，使rhCNTF包涵体几乎完全转化为正确的构象。浓度的稳定操作范围为0.5 - 4.0mg/mL，在此范围内复性产率几乎为100%。与HHP未能将复性产率提高到90%以上的文献报道相比，原因可能在于rhCNTF没有二硫键且是单体蛋白这一结构差异。经过一步阴离子色谱纯化后，rhCNTF的纯度达到95%，总工艺回收率为54.1%。纯化后的rhCNTF与天然蛋白具有相似的结构和体外生物活性。整个过程具有溶解/复性一体化、100%的高复性产率、4mg/mL的高浓度以及简单的色谱法等特点，确保了高生产率。
