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

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 pl...

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Main Author:	Hasmoni, Siti Halimah
Format:	Thesis
Language:	English
Published:	2012
Subjects:	R Medicine (General)
Online Access:	http://eprints.utm.my/id/eprint/34593/1/SitiHalimahHasmoniMFBB2012.pdf
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id	my-utm-ep.34593
record_format	uketd_dc
spelling	my-utm-ep.345932017-09-21T04:13:41Z Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing 2012-11 Hasmoni, Siti Halimah R Medicine (General) 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. 2012-11 Thesis http://eprints.utm.my/id/eprint/34593/ http://eprints.utm.my/id/eprint/34593/1/SitiHalimahHasmoniMFBB2012.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:69748?site_name=Restricted Repository masters Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering Faculty of Biosciences and Medical Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	R Medicine (General)
spellingShingle	R Medicine (General) Hasmoni, Siti Halimah Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing
description	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.
format	Thesis
qualification_level	Master's degree
author	Hasmoni, Siti Halimah
author_facet	Hasmoni, Siti Halimah
author_sort	Hasmoni, Siti Halimah
title	Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing
title_short	Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing
title_full	Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing
title_fullStr	Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing
title_full_unstemmed	Construction of a strep-tag II mutant maltose binding protein for reagentless fluorescence sensing
title_sort	construction of a strep-tag ii mutant maltose binding protein for reagentless fluorescence sensing
granting_institution	Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering
granting_department	Faculty of Biosciences and Medical Engineering
publishDate	2012
url	http://eprints.utm.my/id/eprint/34593/1/SitiHalimahHasmoniMFBB2012.pdf
_version_	1747816212224540672

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

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