Strep-tag II Mutant Maltose-binding Protein for Reagentless Fluorescence Sensing

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Maltose-binding protein (MBP) is crucial for maltose transport and chemotaxis in Gram-negative bacteria.
• MBP undergoes a conformational change upon maltose binding, making it suitable for biosensor applications.
• Existing biosensing methods often require reagents, limiting their practicality.

Purpose of the Study:

• To engineer a mutant maltose-binding protein (MBP) for reagentless fluorescence sensing.
• To develop a sensitive and specific biosensor for maltose detection.
• To investigate the utility of site-directed mutagenesis and fluorescent labeling in MBP-based biosensors.

Main Methods:

• Site-directed mutagenesis was used to introduce a cysteine residue into the malE gene encoding MBP.
• A thiol-specific fluorescent probe (IANBD amide) was attached to the engineered cysteine residue.
• Fluorescence spectroscopy was employed to analyze the ligand-binding properties of the labeled MBP mutant.

Main Results:

• A Strep-tag II-MBP mutant (D95C) was successfully constructed and purified.
• The IANBD-labeled MBP mutant exhibited a significant change in fluorescence intensity upon maltose binding.
• The dissociation constant for maltose was determined to be 7.6±1.75 μM, indicating high affinity.
• The engineered MBP retained its maltose-binding activity.

Conclusions:

• The engineered MBP mutant is suitable for reagentless fluorescence sensing of maltose.
• This approach offers a promising platform for developing novel biosensors.
• The study highlights the potential of protein engineering for creating advanced diagnostic tools.