Engineering periplasmic ligand binding proteins as glucose nanosensors

Diabetes affects over 100 million people worldwide. Better methods for monitoring blood glucose levels are needed for improving disease management. Several labs have previously made glucose nanosensors by modifying members of the periplasmic ligand binding protein superfamily. This minireview summarizes recent developments in constructing new versions of these proteins that are responsive within the physiological range of blood glucose levels, employ new reporter groups, and/or are more robust. These experiments are important steps in the development of novel proteins that have the characteristics needed for an implantable glucose nanosensor for diabetes management: specificity for glucose, rapid response, sensitivity within the physiological range of glucose concentrations, reproducibility, and robustness

A protein switch sensing system for the quantification of sulfate

Protein engineering has generated versatile methods and technologies that have been instrumental in advancements in the fields of sensing, therapeutics, and diagnostics. Herein, we demonstrate the employment of rational design to engineer a unique bioluminescence-based protein switch. A fusion protein switch combines two totally unrelated proteins, with distinct characteristics, in a manner such that the function of one protein is dependent on another. Herein we report a protein switch sensing system by insertion of the sulfate-binding protein (SBP) into the structure of the photoprotein aequorin (AEQ). In the presence of sulfate, SBP undergoes a conformational change bringing the two segments of AEQ together, “turning on” bioluminescence in a dose-dependent fashion, thus allowing quantitative detection of sulfate. A calibration plot was obtained by correlating the amount of bioluminescence generated with the concentration of sulfate present. The switch demonstrated selectivity and reproducibility, and a detection limit of 1.6 × 10 −4 M for sulfate. Moreover, the sensing system was validated by performing sulfate detection in clinical and environmental samples, such as, serum, urine, and tap water. The detection limits and working ranges in all three samples fall within the average normal/recommended sulfate levels in the respective matrices

Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction

Cyclic AMP Receptor Protein−Aequorin Molecular Switch for Cyclic AMP

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an “on/off” signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria. Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP, resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of bioluminescent signal from aequorin. The “on/off” bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as in vivo cyclic AMP detection and/or imaging

Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging

Chapter 13 - Biotechnological Improvements of Bioluminescent Systems

Genetic and biomolecular engineering are two of the leading disciplines in biotechnology that have lead to great advancements in protein engineering. Applications of analytical bioluminescence, such as genetic reporter assays, optical in vivo imaging, and cell viability assays can often be improved by enhancing wild-type bioluminescent systems. The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. Specifically, a wide range of bioluminescent proteins and photoproteins have been engineered that can be utilized in many detection and diagnostic applications. Herein, we focus on the improvements of two of the most commonly studied photoproteins, aequorin and obelin, and their uses in a variety of bioanalytical applications. Techniques such as random mutagenesis, site-directed mutagenesis, bioluminescence resonance energy transfer, and the incorporation of coelenterazine analogues are discussed as ways that have expanded the palette of these designer proteins by altering their emission wavelengths and/or half-lifes. Strategic amino acid substitutions and insertions have been also used to improve luciferase stability in high temperature, extreme pH, and harsh chemical environments, and to customize their kinetic properties and bioluminescence colors. As researchers advance engineering techniques to expand the array of photoproteins, luciferases from fireflies, click beetles, marine organisms and bacteria, their use in bioanalytical applications will continue to grow and it is envisioned that photoproteins and bioluminescent proteins will become as diverse as their fluorescence counterparts

A protein switch sensing system for the quantification of sulfate

Protein engineering has generated versatile methods and technologies that have been instrumental in advancements in the fields of sensing, therapeutics and diagnostics. Herein, we demonstrate the employment of rational design to engineer a unique bioluminescence-based protein switch. A fusion protein switch combines two totally unrelated proteins, with distinct characteristics, in a manner such that the function of one protein is dependent on another. Herein we report a protein switch sensing system by insertion of the sulfate-binding protein (SBP) into the structure of the photoprotein aequorin (AEQ). In the presence of sulfate, SBP undergoes a conformational change bringing the two segments of AEQ together, “turning on” bioluminescence in a dose-dependent fashion, thus allowing quantitative detection of sulfate. A calibration plot was obtained by correlating the amount of bioluminescence generated with the concentration of sulfate present. The switch demonstrated selectivity and reproducibility, and a detection limit of 1.6 × 10(−4) M for sulfate. Moreover, the sensing system was validated by performing sulfate detection in clinical and environmental samples, such as, serum, urine and tap water. The detection limits and working ranges in all three samples fall within the average normal / recommended sulfate levels in the respective matrices