Single chain Fv fragment specific for human GM-CSF: selection and expression using a bacterial expression library.
Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti-human granulocyte macrophage colony-stimulating factor (hGM-CSF) scFv as an immunoassay bio-reagent, utilizing an easily scalable bacterial expression system. For this, the V(H) and V(L) gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre-sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross-reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross-reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM-CSF, measured by surface plasmon resonance, was found to be within the physiological range (K(D) =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM-CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein.
Tapryal
Suman
S
School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Pal Khasa
Yogender
Y
Mukherjee
K J
KJ
D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D051379:Mice; D008807:Mice, Inbred BALB C; D057127:Single-Chain Antibodies / Q000276:immunology*
D057127:Single-Chain Antibodies; D016178:Granulocyte-Macrophage Colony-Stimulating Factor