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Glucose recognition proteins for glucose sensing at physiological concentrations and temperatures.

Joel S, Turner KB, Daunert S - ACS Chem. Biol. (2014)

Bottom Line: The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins.The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability.This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami , 1011 NW 15th Street, Miami, Florida 33136, United States.

ABSTRACT
Advancements in biotechnology have allowed for the preparation of designer proteins with a wide spectrum of unprecedented chemical and physical properties. A variety of chemical and genetic methods can be employed to tailor the protein's properties, including its stability and various functions. Herein, we demonstrate the production of semisynthetic glucose recognition proteins (GRPs) prepared by truncating galactose/glucose binding protein (GBP) of E. coli and expanding the genetic code via global incorporation of unnatural amino acids into the structure of GBP and its fragments. The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins. The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability. These modifications enabled the utilization of the protein for the detection of glucose within physiological concentrations (mM) and temperatures ranging from hypothermia to hyperthermia. This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

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Temperaturestudy of uGRP fiber optic biosensor (A) GRP1-FW and (B) GRP1-FL. Symbolsrepresent (●) 37 °C and (▲) 42.5 °C. Dataare the average of ±1 SD (n = 3). Relative standarddeviations at all concentrations are less than 10%.
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fig7: Temperaturestudy of uGRP fiber optic biosensor (A) GRP1-FW and (B) GRP1-FL. Symbolsrepresent (●) 37 °C and (▲) 42.5 °C. Dataare the average of ±1 SD (n = 3). Relative standarddeviations at all concentrations are less than 10%.

Mentions: To demonstrate the abilityof our genetically engineered uGRPs in sensing applications, we developeda catheter-based biosensor for the continuous detection of glucose.The fluorescence-based biosensor was developed by covalent immobilizationof the MDCC-labeled uGRPs within the UV polymerized acrylamide hydrogelon the tip of an optical fiber. The response of the fiber optic glucosebiosensor was evaluated in standard glucose solutions, in human serum,and in pig blood (Supplememtary information, Figure 1), by monitoring the changes in fluorescence intensityof the probe. The response of the sensor was also studied at differenttemperatures to reflect physiological situations ranging from hypothermiato hyperthermia. The performance of the uGRP1-FW and uGRP1-FL biosensorsin glucose solutions in buffer was also evaluated at 37 and 42.5 °C(Figure 7). The results obtained are thosethat we would expect given the demonstrated thermal stability of theuGRP 1s-FW/FL. The uGRP1-FW has a Tm of41.0 °C, and the sensors incorporating this protein showed verylittle change in fluorescence signal in the presence of differentglucose concentrations at 42.5 °C, thus indicating the loss inactivity of the protein at physiological hyperthermia. In contrast,the uGRP1-FL which has a Tm at 65.3 °C,showed significant fluorescence quenching at both 37 and 42.5 °C.Further, the hydrogel plays an important role in sensing glucose withinthe millimolar detection range. This can be observed for some of theunnatural amino acids incorporated GRPs (SupportingInformation Table 1) whereby although the binding constantis in the micromolar range, the protein still detects glucose in themillimolar ranges (Figure 7). This is becausethe hydrogel functions as a barrier, allowing the diffusion of glucoseto the immobilized protein in the bulk of hydrogel, thus alteringthe detection range and causing the sensor to detect higher levelsof glucose, in this case the targeted physiological millimolar concentrations.Thus, the sensors incorporating designer proteins with enhanced thermalstability retain their binding ability toward glucose and can be employedfor monitoring glucose at phisiological temperatures, from hypothermiato hyperthermia. The response of a hydrogel sensor was also studiedover a period of 3 days (Supporting Information, Figure 2). The hydrogel was deposited on the tip of the fiber,and the response of the sensor to glucose was measured each day. Thesensor was stored at 4 °C in buffer in between measurements.


Glucose recognition proteins for glucose sensing at physiological concentrations and temperatures.

Joel S, Turner KB, Daunert S - ACS Chem. Biol. (2014)

Temperaturestudy of uGRP fiber optic biosensor (A) GRP1-FW and (B) GRP1-FL. Symbolsrepresent (●) 37 °C and (▲) 42.5 °C. Dataare the average of ±1 SD (n = 3). Relative standarddeviations at all concentrations are less than 10%.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215909&req=5

fig7: Temperaturestudy of uGRP fiber optic biosensor (A) GRP1-FW and (B) GRP1-FL. Symbolsrepresent (●) 37 °C and (▲) 42.5 °C. Dataare the average of ±1 SD (n = 3). Relative standarddeviations at all concentrations are less than 10%.
Mentions: To demonstrate the abilityof our genetically engineered uGRPs in sensing applications, we developeda catheter-based biosensor for the continuous detection of glucose.The fluorescence-based biosensor was developed by covalent immobilizationof the MDCC-labeled uGRPs within the UV polymerized acrylamide hydrogelon the tip of an optical fiber. The response of the fiber optic glucosebiosensor was evaluated in standard glucose solutions, in human serum,and in pig blood (Supplememtary information, Figure 1), by monitoring the changes in fluorescence intensityof the probe. The response of the sensor was also studied at differenttemperatures to reflect physiological situations ranging from hypothermiato hyperthermia. The performance of the uGRP1-FW and uGRP1-FL biosensorsin glucose solutions in buffer was also evaluated at 37 and 42.5 °C(Figure 7). The results obtained are thosethat we would expect given the demonstrated thermal stability of theuGRP 1s-FW/FL. The uGRP1-FW has a Tm of41.0 °C, and the sensors incorporating this protein showed verylittle change in fluorescence signal in the presence of differentglucose concentrations at 42.5 °C, thus indicating the loss inactivity of the protein at physiological hyperthermia. In contrast,the uGRP1-FL which has a Tm at 65.3 °C,showed significant fluorescence quenching at both 37 and 42.5 °C.Further, the hydrogel plays an important role in sensing glucose withinthe millimolar detection range. This can be observed for some of theunnatural amino acids incorporated GRPs (SupportingInformation Table 1) whereby although the binding constantis in the micromolar range, the protein still detects glucose in themillimolar ranges (Figure 7). This is becausethe hydrogel functions as a barrier, allowing the diffusion of glucoseto the immobilized protein in the bulk of hydrogel, thus alteringthe detection range and causing the sensor to detect higher levelsof glucose, in this case the targeted physiological millimolar concentrations.Thus, the sensors incorporating designer proteins with enhanced thermalstability retain their binding ability toward glucose and can be employedfor monitoring glucose at phisiological temperatures, from hypothermiato hyperthermia. The response of a hydrogel sensor was also studiedover a period of 3 days (Supporting Information, Figure 2). The hydrogel was deposited on the tip of the fiber,and the response of the sensor to glucose was measured each day. Thesensor was stored at 4 °C in buffer in between measurements.

Bottom Line: The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins.The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability.This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami , 1011 NW 15th Street, Miami, Florida 33136, United States.

ABSTRACT
Advancements in biotechnology have allowed for the preparation of designer proteins with a wide spectrum of unprecedented chemical and physical properties. A variety of chemical and genetic methods can be employed to tailor the protein's properties, including its stability and various functions. Herein, we demonstrate the production of semisynthetic glucose recognition proteins (GRPs) prepared by truncating galactose/glucose binding protein (GBP) of E. coli and expanding the genetic code via global incorporation of unnatural amino acids into the structure of GBP and its fragments. The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins. The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability. These modifications enabled the utilization of the protein for the detection of glucose within physiological concentrations (mM) and temperatures ranging from hypothermia to hyperthermia. This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

Show MeSH
Related in: MedlinePlus