Negative effects of a non-consensus Cys residue of a single‑chain antibody fragment (scFv)

AGUILAR, MA. FERNANDA; FORNO, GUILLERMINA; ETCHEVERRIGARAY, MARINA; ATTALLAH, CAROLINA; OGGERO, MARCOS

Mar del Plata

Congreso; 51º Reunión anual de la Sociedad Argentina de Bioquímica y Biología Molecular LI Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

scFv(s) have the potential to replace monoclonal antibodies (mAbs) in several applications. The aim of this work was to obtain a scFv anti-recombinant human follicle stimulating hormone (rhFSH). Besides, given the attained sequence, the effect of two contiguous Cys residues on the antigen interaction was analyzed. A pool of heavy and light variable fragments (VH-VL) was constructed from an anti-rhFSH hybridoma-extracted RNA. VH and VL fragments were identified by sequencing and bioinformatics. Then, the scFv was constructed by splicing of VH and VL nucleotide sequences through a linker. The scFv was expressed in the periplasmic space of E. coli BL21(DE3) RosettaTM cells and it was extracted by osmotic shock. scFv binding activity to rhFSH was determined by ELISA assays. The scFv sequencing revealed the presence of two contiguous Cys residues in VL fragment. In order to study the impact of these residues, Cys-by-Ser mutations were carried out considering their similar physicochemical properties and the absence of the sulfhydryl group in Ser. Thus, C87S and C88S mutants were constructed by site-directed mutagenesis. The mutation of Cys-VL88 confers a non scFv binding to rhFSH and the mutation of Cys-VL87 produced an increase of the scFv anti-rhFSH affinity. These results led to the conclusion that Cys‑VL87 would cause the structural destabilization of the scFv to bind to the hormone.