Fusion protein of CDR mimetic peptide with Fc inhibit TNF-alpha induced cytotoxicity.
The variable regions of antibodies play central roles in the binding with antigens. Based on the model of a tumour necrosis factor-alpha (TNF-alpha) neutralizing monoclonal antibody (named as Z12) with TNF-alpha, heavy chain CDR2 (HCDR2) and light chain CDR3 (LCDR3) of Z12 were found to be the most responsible to bind with TNF-alpha. A mimetic peptide (PT) was designed based on the sequence derived from HCDR2 and LCDR3. Fusion protein PT-Fc was constructed by linking PT with Fc of human IgG1 through a flexible linker (GGGGGS). The primary structural characteristics of Fc and PT-Fc were analyzed, including the flexibility, hydrophilicity and epitopes. It was demonstrated that PT and Fc in the fusion protein possessed bio-function properly and non-interfering with each other. Furthermore, PT-Fc was expressed in Escherichia coli by fusion with thioredoxin (Trx). After trx-PT-Fc was cleaved with recombinant enterokinase, PT-Fc was obtained. The results of in vitro cytotoxic assays showed that both PT and PT-Fc could efficiently inhibit TNF-alpha induced apoptosis on L929 cells. At the same micromole concentration, the inhibition activity of PT-Fc was significantly higher than PT.
Qin
Weisong
W
Institute of Basic Medical Sciences, P.O. Box 130 (3) Taiping Road, 100850 Beijing, PR China.
Feng
Jiannan
J
Li
Yan
Y
Lin
Zhou
Z
Shen
Beifen
B
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D022801:Complementarity Determining Regions; D000939:Epitopes; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D051379:Mice; D018716:Molecular Mimicry; D010446:Peptide Fragments; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D022801:Complementarity Determining Regions; D000939:Epitopes; D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha