Abstract:
:Deoxynivalenol (DON), a mycotoxin produced by several Fusarium species, is a worldwide contaminant of food and feedstuffs. The DON-specific single-chain variable fragment (scFv) antibody was produced in recombinant Escherichia coli. The variable regions of the heavy chain (V(H)) and light chain (V(L)) cloned from the hybridoma 3G7 were connected with a flexible linker using an overlap extension polymerase chain reaction. Nucleotide sequence analysis revealed that the anti-DON V(H) was a member of the V(H) III gene family IA subgroup and the V(L) gene belonged to the Vlambda gene family II subgroup. Extensive efforts to express the functional scFv antibody in E. coli have been made by using gene fusion and chaperone coexpression. Coexpression of the molecular chaperones (DnaK-DnaJ-GrpE) allowed soluble expression of the scFv. The scFv antibody fused with hexahistidine residues at the C-terminus was purified by immobilized metal affinity chromatography (IMAC). Soluble scFv antibody produced in this manner was characterized for its antigen-binding characteristics. Its biological affinity as antibody was measured by surface plasmon resonance (SPR) analysis and proved to be significant but weaker than that of the whole anti-DON mAb.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Choi GH,Lee DH,Min WK,Cho YJ,Kweon DH,Son DH,Park K,Seo JHdoi
10.1016/j.pep.2003.12.008keywords:
subject
Has Abstractpub_date
2004-05-01 00:00:00pages
84-92issue
1eissn
1046-5928issn
1096-0279pii
S1046592804000105journal_volume
35pub_type
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