Modular protein switches derived from antibody mimetic proteins

Abstract

Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms.

Proteome editing using engineered proteins that hijack cellular quality control machinery

“…This could be accomplished by generating stimuli‐sensitive switches that effectively control degrader activity. Along these lines, Ostermeier and coworkers have described methods for creating enzyme‐binding protein hybrids whose enzymatic activity is activated only in the presence of antigen . Another option would be the creation of light‐inducible switches that could be conditionally activated in precise areas of the body, preferably using longer wavelengths of light.…”

Quantitative and synthetic biology approaches to combat bacterial pathogens

“…For instance, the examples listed above focus on sensing small molecules. The sensing capability can be further expanded by incorporating additional sensing and actuating functions, such as those based on engineered protein switches [51,52]. Moreover, instead of storing the sensory information by regulating gene expression, the memory recorded in DNA molecules in single cells through stimuli-responsive genome editing can provide relatively more stable memory storage [53,54].…”

Possibilities of radionuclide visualization of HER2/neu-positive breast cancer using a radiopharmaceutical based on recombinant targeting molecules DARPin9_29

“…На данном этапе развития молекулярных и генетических технологий для этих целей используются структуры, получившие название «альтернативных каркасных белков» [9,10]. Основные преимущества таких молекулярных конструкций обусловлены их связыванием исключительно с таргетным антигеном для специфической локализации, отсутствием иммуногенности, стабильностью и возможностью быстрой химической модификации для проведения процессов мечения [11]. Существенным для медицинской визуализации являются также скорость связывания с мишенью и быстрое удаление несвязавшихся молекул из организма пациента для достижения высокого качества визуализации опухоли и сокращения временного промежутка между инъекцией и началом исследования [12].…”