149
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_10, © Springer Science+Business Media LLC 2017
Chapter 10
Creation of Antigen-Dependent β-Lactamase Fusion
Protein Tethered by Circularly Permuted Antibody
Variable Domains
Hiroto Iwai, Miki Kojima-Misaizu, Jinhua Dong, and Hiroshi Ueda
Abstract
Antibody-based molecular switches that are able to recognize a range of exogenous antigens can be highly
useful as a versatile biosensor. However, regulating the catalytic activity of enzymes by antibodies is still
hard to achieve. Here, we describe a design method of unique antibody variable region Fv introduced with
two circular permutations, called Clampbody. By tethering the Clampbody to a circularly permuted
TEM-1 β-lactamase (BLA), we successfully constructed a genetically encoded molecular switch Cbody- cpBLA
that shows antigen-dependent catalytic activity.
Key words TEM-1 β-lactamase, Antibody variable region, Circular permutation, Allosteric regulation,
Immunosensor
1 Introduction
Regulating the catalytic activity of enzymes by exogenous substances is potentially useful for the detection of biomarkers, environmental pollutants, and for enzyme-based prodrug therapies
[1, 2]. To create the molecular switches that can recognize a wide
variety of molecules, methods to control the catalytic activity of an
enzyme by means of antibody domains are considered highly
important [3].
To date, there have been a number of fusion proteins of antibody domains and enzymes whose catalytic activity was regulated
by the binding antigen-dependent binding to their cognate antibody [4, 5]. Recently, we reported a fusion protein consisting of a
circularly permuted TEM-1 β-lactamase (cpBLA) and Fv, called
Fv-cpBLA [6]. This protein switch was made on a cpBLA that had
been successfully used to make a maltose-dependent switch when
fused with a circularly permuted maltose-binding protein (cpMBP)
[7–9]. Fv-cpBLA showed antigen-dependent catalytic activation
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_10, © Springer Science+Business Media LLC 2017
Chapter 10
Creation of Antigen-Dependent β-Lactamase Fusion
Protein Tethered by Circularly Permuted Antibody
Variable Domains
Hiroto Iwai, Miki Kojima-Misaizu, Jinhua Dong, and Hiroshi Ueda
Abstract
Antibody-based molecular switches that are able to recognize a range of exogenous antigens can be highly
useful as a versatile biosensor. However, regulating the catalytic activity of enzymes by antibodies is still
hard to achieve. Here, we describe a design method of unique antibody variable region Fv introduced with
two circular permutations, called Clampbody. By tethering the Clampbody to a circularly permuted
TEM-1 β-lactamase (BLA), we successfully constructed a genetically encoded molecular switch Cbody- cpBLA
that shows antigen-dependent catalytic activity.
Key words TEM-1 β-lactamase, Antibody variable region, Circular permutation, Allosteric regulation,
Immunosensor
1 Introduction
Regulating the catalytic activity of enzymes by exogenous substances is potentially useful for the detection of biomarkers, environmental pollutants, and for enzyme-based prodrug therapies
[1, 2]. To create the molecular switches that can recognize a wide
variety of molecules, methods to control the catalytic activity of an
enzyme by means of antibody domains are considered highly
important [3].
To date, there have been a number of fusion proteins of antibody domains and enzymes whose catalytic activity was regulated
by the binding antigen-dependent binding to their cognate antibody [4, 5]. Recently, we reported a fusion protein consisting of a
circularly permuted TEM-1 β-lactamase (cpBLA) and Fv, called
Fv-cpBLA [6]. This protein switch was made on a cpBLA that had
been successfully used to make a maltose-dependent switch when
fused with a circularly permuted maltose-binding protein (cpMBP)
[7–9]. Fv-cpBLA showed antigen-dependent catalytic activation