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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
2017-11-01
Source PublicationBIOCONJUGATE CHEMISTRY
ISSN1043-1802
Volume28Issue:11Pages:2841-2848
Abstract

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.

SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
DOI10.1021/acs.bioconjchem.7600531
Indexed BySCI
Language英语
WOS KeywordProtein Pegylation ; N-ethylmaleimide ; Inclusion-bodies ; Denaturation ; Gelation ; Cntf
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS SubjectBiochemical Research Methods ; Biochemistry & Molecular Biology ; Chemistry, Multidisciplinary ; Chemistry, Organic
Funding OrganizationNational Natural Science Foundation of China(21576267) ; Beijing Natural Science Foundation(2162041) ; Major State Basic Research Development Program of China(2013CB733604) ; National Key Laboratory of Biochemical Engineering(2014KF-05)
WOS IDWOS:000415785600019
Citation statistics
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/23389
Collection生化工程国家重点实验室
Affiliation1.Chinese Acad Sci, State Key Lab Biochem Engn, Inst Proc Engn, 1 North Second St, Beijing 100190, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Jiangsu Natl Synerget Innovat Ctr Adv Mat SICAM, Nanjing 210023, Jiangsu, Peoples R China
Recommended Citation
GB/T 7714
Wang, Qi,Zhang, Chun,Guo, Fangxia,et al. 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[J]. BIOCONJUGATE CHEMISTRY,2017,28(11):2841-2848.
APA Wang, Qi,Zhang, Chun,Guo, Fangxia,Li, Zenglan,Liu, Yongdong,&Su, Zhiguo.(2017).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.BIOCONJUGATE CHEMISTRY,28(11),2841-2848.
MLA Wang, Qi,et al."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".BIOCONJUGATE CHEMISTRY 28.11(2017):2841-2848.
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