Universiti Teknologi Malaysia Institutional Repository

Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing

Hasmoni, Siti Halimah (2012) Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing. Masters thesis, Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering.

[img]
Preview
PDF
630kB

Official URL: http://dms.library.utm.my:8080/vital/access/manage...

Abstract

Maltose binding protein (MBP) changes its conformational structure upon its ligand binding.This molecular recognition element that transduces a ligand-binding event into a physical one make MBP an ideal candidate for reagentless fluorescence sensing. MBP gene, (malE) was amplified from a pMaL-C4x plasmid vector and was fused to a Strep-Tag II pET-51b(+) vector. Strep-Tag II is a tag that will enable the MBP to be unidirectionally immobilized on solid supports. A cysteine mutant of the MBP was constructed by inverse PCR and the recombinant protein fusion was then purified by affinity purification using Strep-Tactin resin. To sense maltose binding, an environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group. The tagged mutant MBP (D95C) was successfully generated and the protein was successfully purified with the expected molecular size of ~42 kDa observed on the SDS PAGE. The fluorescence measurements of the IANBD labeled of tagged mutant MBP (Strep-Tag II D95C) in the solution phase, showed an appreciable change in fluorescence intensity with dissociation constant, (Kd) of 7.6 ± 1.75 µM. Nonetheless, it could retain its ligand binding activity towards maltose. However, immobilization of Strep-Tag II D95C on solid surface suffered some limitation with the Strep-Tactin coated microwell plates because it did not give any dependable results to support the ligand binding activity of the site directed immobilized protein. Thus, this engineered mutant MBP (Strep-Tag II fused D95C) could be potentially developed for biosensor application with further improvement in protein immobilization method.

Item Type:Thesis (Masters)
Additional Information:Thesis (Sarjana Sains (Biosains)) - Universiti Teknologi Malaysia, 2012; Supervisor : Dr. Shafinaz Shahir, Prof. Dr. Anthony Cass
Uncontrolled Keywords:biosensors, biosensing techniques
Subjects:R Medicine > R Medicine (General)
Divisions:Biosciences and Medical Engineering
ID Code:34593
Deposited By: Kamariah Mohamed Jong
Deposited On:20 Feb 2014 10:03
Last Modified:21 Sep 2017 12:13

Repository Staff Only: item control page