Document Detail


Covalently dimerized Camelidae antihuman TNFa single-domain antibodies expressed in yeast Pichia pastoris show superior neutralizing activity.
MedLine Citation:
PMID:  23324801     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Antagonists of tumor necrosis factor alpha (TNFa) have revolutionized the treatment of selected inflammatory diseases. Recombination Camelidae variable heavy-chain domain-only TNFa antibodies (anti-TNF-V(H)H) have been developed to antagonize the action of human and murine TNFa. Here, we describe a strategy to obtain functional covalent dimer anti-TNF-V(H)H molecules with the C-terminal fusion of human IgG1 Fc domain named anti-TNF-V(H)H-Fc. The resulting fusion proteins were separately expressed by use of the pET28a vector in Escherichia coli ((Ec)) strain BL21 and the pPICZaA vector in Pichia pastoris ((Pp)) strain GS115, then purified by protein A affinity resin. Fc-engineered anti-(Ec)TNF-V(H)H-Fc was about 40 kDa and anti-(Pp)TNF-V(H)H-Fc was about 43 kDa. Monomeric V(H)H was also cloned and expressed in E. coli strain BL21, with the molecular weight of about 18 kDa. Enzyme-linked immunosorbent assay and L929 cell cytotoxicity assay demonstrated that the fusion protein anti-(Pp)TNF-V(H)H-Fc blocked TNFa activity more effectively than either anti-(Ec)TNF-V(H)H-Fc or monomeric anti-(Ec)TNF-V(H)H protein. We suggest that efficient disulfide bond formation using the P. pastoris expression system improves the covalent dimer anti-TNF-V(H)H-Fc neutralizing activity.
Authors:
Xuemei Ji; Wuguang Lu; Huiting Zhou; Dongju Han; Lin Yang; Haitao Wu; Jianfeng Li; Hongyan Liu; Jie Zhang; Peng Cao; Shuangquan Zhang
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-17
Journal Detail:
Title:  Applied microbiology and biotechnology     Volume:  -     ISSN:  1432-0614     ISO Abbreviation:  Appl. Microbiol. Biotechnol.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-17     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8406612     Medline TA:  Appl Microbiol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Jiang Su Province Key Laboratory for Molecular and Medical Biotechnology, Life Science College, Nanjing Normal University, Nanjing, 210046, Jiangsu, China.
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