Cloning, expression, and characterization of single-chain variable fragment antibody against mycotoxin deoxynivalenol in recombinant Escherichia coli.
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.
Choi
Gyu-Ho
GH
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea.
Lee
Dae-Hee
DH
Min
Won-Ki
WK
Cho
Young-Jin
YJ
Kweon
Dae-Hyuk
DH
Son
Dong-Hwa
DH
Park
Kyungmoon
K
Seo
Jin-Ho
JH
D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000737:chemistry*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment; D014255:Trichothecenes / Q000276:immunology*
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D018832:Molecular Chaperones; D009183:Mycotoxins; D011993:Recombinant Fusion Proteins; D014255:Trichothecenes; C007262:deoxynivalenol