Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus: bacterial expression and refolding.
Single chain antibodies (ScFvs) are heavy and light chain variable domains connected by an artificial linker. Because of their smaller size, ScFvs show improved tissue penetration in vivo and reduced immunogenicity, making them ideal for therapeutic applications. We have cloned a ScFv against western equine encephalitis (WEE) using rDNA technology. The ScFv was generated from a hybridoma cell line (11D2) specific to the WEE virus E1 glycoprotein and is arranged in the V(L)-V(H) orientation with a (gly(4)ser)(3) linker. This ScFv was engineered successfully with a biotin mimic tag (11 amino acid peptide) and cloned in the pET22b+ expression vector. The ScFv was expressed as a approximately 32kDa protein in Escherichia coli as inclusion bodies, with an estimated yield of 20-40 mg/l. Different refolding protocols were used to solubilise the inclusion bodies. Most of the functional ScFv was generated when the inclusion bodies were solubilized in a detergent, air oxidised in the presence of CuSO(4) and then denatured in urea buffer in comparison to other protocols. The product was renatured finally in Tris arginine buffer (pH 8.0). Refolded protein was dialysed against phosphate buffer saline (PBS) (pH 7.3) to remove the Tris and arginine. Our refolding protocol generated up to a 50% yield of soluble protein, which retained antigen-binding activity with whole inactivated WEE virus as demonstrated by ELISA and Western blot analysis. This 11D2-biotin mimic ScFv complexed with streptavidin horseradish peroxidase (St-HRPO) will be useful as a detector reagent in the ultrasensitive ELISA detection of WEE virus antigen.
Das
D
D
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada T6G 2N8.
Kriangkum
J
J
Nagata
L P
LP
Fulton
R E
RE
Suresh
M R
MR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000235:genetics; D004667:Encephalitis Virus, Western Equine / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000032:analysis
D004275:DNA, Ribosomal; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D001710:Biotin