197
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_13, © Springer Science+Business Media LLC 2017
Chapter 13
Engineering and Characterizing Synthetic Protease
Sensors and Switches
Viktor Stein and Kirill Alexandrov
Abstract
Proteases are finding an increasing number of applications as molecular tools and reporters in biotechnology
and basic research. Proteases are also increasingly incorporated into synthetic genetic signaling circuits
equipping cells with tailored new functions. In the majority of cases however, proteases are employed in
constitutively active forms which limits their utility and application as molecular sensors. The following
chapter provides a detailed experimental protocol for converting constitutively active proteases into regulated
protease receptors. Such receptors can potentially sense, transduce, and amplify any molecular input,
thereby opening up a range of new applications in basic research, biotechnology, and synthetic biology.
Key words Protein switches, Protein engineering, Proteases, Diagnostic reagents
1 Introduction
Proteases constitute one of the most abundant classes of enzymes
that execute key physiological functions across all kingdoms of
life [1]. Proteases have evolved to function inside as well as outside
cellular environments taking on diverse roles such as regulating cell
death, controlling blood coagulation, digesting nutrients, and
remodeling the extracellular environment. Chemically, proteases
catalyze the irreversible cleavage of a peptide bond. Their substrate
specificity ranges from peptide motives as short as two amino acids,
to complex tertiary interactions that are mediated by structurally
distinct protein domains. Given the central importance of proteases to many physiological processes and the irreversible nature of
the proteolytic cleavage, protease function is frequently tightly
regulated. One common mechanism relies on the expression of
inactive zymogens that require additional posttranslational processing by an activating protease that cleaves off an active sitedirected inhibitor or triggers complex conformational
rearrangements allowing a protease to transition into a catalytically
active conformation.
1.1 Proteases
as Versatile Tools
in Biotechnology
and Basic Research
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_13, © Springer Science+Business Media LLC 2017
Chapter 13
Engineering and Characterizing Synthetic Protease
Sensors and Switches
Viktor Stein and Kirill Alexandrov
Abstract
Proteases are finding an increasing number of applications as molecular tools and reporters in biotechnology
and basic research. Proteases are also increasingly incorporated into synthetic genetic signaling circuits
equipping cells with tailored new functions. In the majority of cases however, proteases are employed in
constitutively active forms which limits their utility and application as molecular sensors. The following
chapter provides a detailed experimental protocol for converting constitutively active proteases into regulated
protease receptors. Such receptors can potentially sense, transduce, and amplify any molecular input,
thereby opening up a range of new applications in basic research, biotechnology, and synthetic biology.
Key words Protein switches, Protein engineering, Proteases, Diagnostic reagents
1 Introduction
Proteases constitute one of the most abundant classes of enzymes
that execute key physiological functions across all kingdoms of
life [1]. Proteases have evolved to function inside as well as outside
cellular environments taking on diverse roles such as regulating cell
death, controlling blood coagulation, digesting nutrients, and
remodeling the extracellular environment. Chemically, proteases
catalyze the irreversible cleavage of a peptide bond. Their substrate
specificity ranges from peptide motives as short as two amino acids,
to complex tertiary interactions that are mediated by structurally
distinct protein domains. Given the central importance of proteases to many physiological processes and the irreversible nature of
the proteolytic cleavage, protease function is frequently tightly
regulated. One common mechanism relies on the expression of
inactive zymogens that require additional posttranslational processing by an activating protease that cleaves off an active sitedirected inhibitor or triggers complex conformational
rearrangements allowing a protease to transition into a catalytically
active conformation.
1.1 Proteases
as Versatile Tools
in Biotechnology
and Basic Research