Email this article Recombinant Production of Horseradish Peroxidase Conjugates with Fab Antibodies in Pichia pastoris for Analytical Applications
- Authors: Koliasnikov OV1, Grigorenko VG2, Egorov AM2, Lange S3, Schmid RD3
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Affiliations:
- Kolmogorov Advanced Education and Science Center, Lomonosov Moscow State University
- Lomonosov Moscow State University
- Institute of Technical Biochemistry, University of Stuttgart
- Issue: Vol 3, No 3 (2011)
- Pages: 85-92
- Section: Articles
- Submitted: 17.01.2020
- Published: 15.09.2011
- URL: https://actanaturae.ru/2075-8251/article/view/10697
- DOI: https://doi.org/10.32607/20758251-2011-3-3-85-92
- ID: 10697
Cite item
Abstract
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.
Keywords
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)
Full Text
Recombinant Production of Horseradish Peroxidase Conjugates with Fab Antibodies in Pichia pastoris for Analytical Applications×
About the authors
O V Koliasnikov
Kolmogorov Advanced Education and Science Center, Lomonosov Moscow State UniversityDepartment of Chemistry
V G Grigorenko
Lomonosov Moscow State University
Email: vitaly@immunotek.ru
Department of Chemistry
A M Egorov
Lomonosov Moscow State UniversityDepartment of Chemistry
S Lange
Institute of Technical Biochemistry, University of Stuttgart
R D Schmid
Institute of Technical Biochemistry, University of Stuttgart
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