Recombinant Production of Horseradish Peroxidase Conjugates with Fab Antibodies in Pichia pastoris for Analytical Applications

Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) with Fab fragments of antibodies against atrazine. The resulting genetic construction allows us to switch to any other antibody sequence, via the simple re-cloning of variable parts and an additional reporter enzyme. Conjugates were successfully produced in the Pichia pastoris methylotrophic yeast expression system. The target activity of the conjugates (both enzymatic and antigen-binding) has been demonstrated by ELISA method.

horseradish peroxidase, antibodies, recombinant conjugates, Pichia pastoris expression. ABBREVIATIONS HRP - horseradish peroxidase, ELISA - enzyme-linked immunosorbent assay, BSA - bovine serum albumin, PCR - polymerase chain reaction, TMB - 3,3’,5,5’-tetramethylbenzidine, ABTS - 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)