High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.
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.
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.
Qi
Q
Wang
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. Electronic address: ydliu@ipe.ac.cn.
Yongdong
Y
Liu
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.
Chun
C
Zhang
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.
Fangxia
F
Guo
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.
Cui
C
Feng
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.
Xiunan
X
Li
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.
Hong
H
Shi
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. Electronic address: zgsu@ipe.ac.cn.
Zhiguo
Z
Su
D020934:Ciliary Neurotrophic Factor / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D006874:Hydrostatic Pressure; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D020934:Ciliary Neurotrophic Factor; D011994:Recombinant Proteins
High hydrostatic pressure; Hydrophobic aggregation; Inclusion bodies; Refolding; rhCNTF