Comparative assessment of the VH-VL and VL-VH orientations of single-chain variable fragments of scorpion toxin-neutralizing antibodies

Lidia Riaño-Umbarila; Vianey Margarita Rojas-Trejo; José Alberto Romero-Moreno; Miguel Costas; Irving Utrera-Espíndola; Timoteo Olamendi-Portugal; Lourival D Possani; Baltazar Becerril

doi:10.1016/j.molimm.2020.04.015

Comparative assessment of the VH-VL and VL-VH orientations of single-chain variable fragments of scorpion toxin-neutralizing antibodies

Mol Immunol. 2020 Apr 29:122:141-147. doi: 10.1016/j.molimm.2020.04.015. Online ahead of print.

Authors

Lidia Riaño-Umbarila¹, Vianey Margarita Rojas-Trejo², José Alberto Romero-Moreno², Miguel Costas³, Irving Utrera-Espíndola², Timoteo Olamendi-Portugal², Lourival D Possani², Baltazar Becerril⁴

Affiliations

¹ Cátedra CONACYT, Instituto de Biotecnología-Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, 62250, Mexico. Electronic address: umbarila@ibt.unam.mx.
² Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62250, Mexico.
³ Laboratorio de Bio-Fisicoquímica, Departamento de Fisicoquímica, Facultad de Química, Cd. Universitaria, UNAM, Ciudad de México, 04510, Mexico.
⁴ Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos, 62250, Mexico. Electronic address: baltazar@ibt.unam.mx.

PMID: 32361416
DOI: 10.1016/j.molimm.2020.04.015

Abstract

The present study evaluated the effect of the change in the orientation of the VH-VL variable domains to VL-VH on the physicochemical and functional properties of two scorpion toxin-neutralizing scFvs. The results showed that the level of expression of proteins obtained from the periplasm of E. coli is the factor mainly affected, either with an increase or decrease in the amount of protein recovered. Likewise, the functional recognition activity in the presence of a denaturing agent showed slight variations in the two orientations. In contrast, recognition and biological activity (neutralizing capacity) are maintained. At the interaction level, the change marginally modified the kinetic association and dissociation constants without significantly modifying the value of the affinity constants. Similarly, it was observed that the thermodynamic stability of the proteins did not show significant variations either. These results contrast with some reports of the effect of changing the orientation of domains, suggesting that it is not possible to predict which orientation of the variable domains of an scFv is more favorable or if they are equivalent, as in the case of scFvs previously matured by directed evolution techniques.

Keywords: Expression; Neutralization; Scorpion toxins; Stability; VL-VH orientations; scFv.