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Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A Case Study for the Site-Specific Attachment of Polyethylene Glycol (PEGylation) of Recombinant Human Ciliary Neurotrophic Factor.

  • Wang, Qi1, 2
  • Zhang, Chun1
  • Guo, Fangxia1, 2
  • Li, Zenglan1, 2
  • Liu, Yongdong1
  • Su, Zhiguo1, 3
  • 1 State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences , 1 North Second Street, Zhong-Guan Village, Beijing 100190, PR China. , (China)
  • 2 University of Chinese Academy of Sciences , Beijing 100049, PR China. , (China)
  • 3 Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM) , Nanjing, 210023, PR China. , (China)
Published Article
Bioconjugate Chemistry
American Chemical Society
Publication Date
Nov 15, 2017
DOI: 10.1021/acs.bioconjchem.7b00531
PMID: 29053917


In this paper, we reported a novel strategy for the site-specific attachment of polyethylene glycol (PEGylation) of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was monomethoxy-polyethylene glycol-maleimide (mPEG-MAL). PEGylation with mPEG (40 kDa)-MAL at pH 7.0 under normal pressure for 5 h achieved a less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: (1) high-hydrostatic-pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG-MAL), while the conventional process needs a much-higher ratio. (2) Short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. (3) The reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by a factor of three from pH 7.0 to 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software, and we found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was the facilitation of target-site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated that the HHPP product was comparable to the PEGylated rhCNTF prepared through a conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules.

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