title: Strep-tag II Mutant Maltose-binding Protein for Reagentless Fluorescence Sensing.
abstract: Maltose-binding protein (MBP) is a periplasmic binding protein found in Gram negative bacteria. MBP is involved in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1-4)-glucosidically linked linear glucose polymers and α(1-4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP changes its conformation from an open to a closed form. This molecular recognition-transducing a ligand-binding event into a physical one-renders MBP an ideal candidate for biosensor development. Here, we describe the construction of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified. A cysteine residue was introduced by site-directed mutagenesis to ensure a single label attachment at a specific site with a thiol-specific fluorescent probe. An environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular size, ∼42 kDa). The fluorescence measurements of the IANBD-labelled Strep-tag II-D95C in the solution phase showed an appreciable change in fluorescence intensity (dissociation constant, 7.6±1.75 μM). Our mutant MBP retains maltose-binding activity and is suitable for reagentless fluorescence sensing.
authors.forename: Siti Halimah
authors.initials: SH
authors.lastname: Hasmoni
authors.forename: Goh Kian
authors.initials: GK
authors.lastname: Mau
authors.forename: Saiful Anuar
authors.initials: SA
authors.lastname: Karsani
authors.forename: Anthony
authors.initials: A
authors.lastname: Cass
authors.forename: Shafinaz
authors.initials: S
authors.lastname: Shahir
keywords: D95C; Fluorescence Sensing; Maltose-binding Protein; Strep-tag II