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

Date Published: , 2011

Publisher: A.I. Gordeyev

Author(s): O.V. Koliasnikov, V.G. Grigorenko, A.M. Egorov, S. Lange, R.D. Schmid.

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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) withFabfragments 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 thePichia
pastorismethylotrophic yeast expression system. The target activity
of the conjugates (both enzymatic and antigen-binding) has been demonstrated by
ELISA method.

Partial Text

Enzyme immunoassays for the detection and quantitative analysis of various substances
are based on coupling of marker enzymes such as horseradish peroxidase (HRP, EC
1.11.1.7]) with antigens or antibodies.
However, all major approaches used for the chemical conjugation of proteins and
haptens result in the partial inactivation of the enzyme and conjugate
heterogeneity, which affects the specificity and sensitivity of the ELISA. Genetic
engineering can be used to obtain recombinant conjugates of proteins with antigens
or antibodies. Such conjugates present a number of advantages; firstly, they have a
homogenous composition, secondly, they possess 1 : 1 stoichiometry, thirdly they
retain the functional activities of both the marker protein and that of the
antigen/antibody, in addition to the reproducibility and the fact that they are
relatively simple to produce. Recombinant conjugates of antibodies with alkaline
phosphatase [1–3], luciferase [4], and peroxidase Arthromyces
ramosus [5] were obtained
earlier.

Reagents

Recombinant conjugates are chimeric proteins combining the structural components of
both marker enzymes and an antigen/antibody. The use of modern approaches has almost
solved the problem of obtaining such recombinant enzymes as alkaline phosphatase,
β-galactosidase, luciferase, and horseradish peroxidase that are used as
markers in the ELISA methods. However, the production of recombinant conjugates is
an appreciably complicated task, since it remains thus far impossible to reliably
predict the structure of the desired conjugate; hence, loss of the functional
activity of both the marker enzyme and antigen is possible, due to the incorrect
folding of two components.

The possibility of using a recombinant, functionally active HRP (as a marker enzyme)
conjugated with Fab fragments of the antibody against atrazine was shown for the
first time. In the present study, recombinant conjugates were obtained in which the
Fab fragment of an antibody is bound both to the N- and the C- terminuses of the
marker enzyme. Both these variants manifest immunological and catalytic
activity.

 

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