Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.
A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time.
['Piscitelli|Alessandra|A|', 'Pennacchio|Anna|A|', 'Cicatiello|Paola|P|', 'Politi|Jane|J|', 'De Stefano|Luca|L|', 'Giardina|Paola|P|']
D000327:Adsorption D000818:Animals D015374:Biosensing Techniques D003001:Cloning, Molecular D049452:Green Fluorescent Proteins D006801:Humans D037402:Hydrozoa D055767:Immobilized Proteins D057230:Limit of Detection D010843:Pichia D011137:Polystyrenes D011993:Recombinant Fusion Proteins D013050:Spectrometry, Fluorescence D013917:Thrombin
Class I hydrophobin Protease Amyloid fibrils Yeast heterologous expression