Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography

Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography

The purification of protein using IMAC can be influenced by imidazole concentration and type of metal ion. Different type of metal ion and different imidazole concentration will give different result of purity and yield for target protein. The strength of binding between histidine- tagged protein an...

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Main Author: Insyirah, Samsuddin
Format: Undergraduates Project Papers
Language:English
Published: 2014
Subjects:
QP Physiology
Online Access:http://umpir.ump.edu.my/id/eprint/8853/
http://umpir.ump.edu.my/id/eprint/8853/
http://umpir.ump.edu.my/id/eprint/8853/1/CD8547.pdf
id ump-8853
recordtype eprints
spelling ump-88532015-10-26T06:25:08Z http://umpir.ump.edu.my/id/eprint/8853/ Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography Insyirah, Samsuddin QP Physiology The purification of protein using IMAC can be influenced by imidazole concentration and type of metal ion. Different type of metal ion and different imidazole concentration will give different result of purity and yield for target protein. The strength of binding between histidine- tagged protein and metal ion is varies depending on the type of protein as well as the ion used. Imidazole is one of the competitive agents that are effective at displacing protein. Because of this, imidazole is one of the best agent to be used an eluent. Imidazole at low concentration is commonly use to minimise binding of host cell protein and at somewhat higher concentration, imidazole may also decrease the binding of histidine tagged- protein. This paper presents the effect of imidazole concentration and type of metal ion studies on the purification of recombinant green fluorescent protein (GFP) using an affinity chromatography. Escherichia coli (E.coli) BL21 (DE3) strain carrying the pRSETGFP plasmid encoding the GFP was grown in Luria-Bertani (LB) broth at 300C and 200 rpm for 16 hours. An induction of protein expression, Isopropyl β-ᴅ-1- thiogalactopyranoside when the cell density (OD600) reaches 0.6-0.8 enhanced the yield of functional GFP production. After harvesting, the cell suspension was disrupted using freeze and thaw method. Clarified GFP was then filter using nylon filter before being purified using a HisTrapTM FF 1 ml column (GE healthcare). The column was charged with different metal ion (0.1 M NiSO4 or CuSO4) and the concentration of imidazole at elution buffer was varied (100, 200, 300, 400, 500, 600 mM). For protein analysis, the sample was analysed using Lowry method for total protein determination, while amount of GFP was quantified using gel-based imaging method. This method has resulted in 95% purity at 500 mM imidazole concentration and 93% recovery of GFP at 300 mM imidazole concentration when the column was charged with Ni(II) ion while 89% purity at 500 mM imidazole concentration and 95 % recovery at 400 mM at imidazole concentration of GFP when the column was charged with Cu(II) ion 2014-01 Undergraduates Project Papers NonPeerReviewed application/pdf en http://umpir.ump.edu.my/id/eprint/8853/1/CD8547.pdf Insyirah, Samsuddin (2014) Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography. Faculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang. http://iportal.ump.edu.my/lib/item?id=chamo:84869&theme=UMP2
repository_type Digital Repository
institution_category Local University
institution Universiti Malaysia Pahang
building UMP Institutional Repository
collection Online Access
language English
topic QP Physiology
spellingShingle QP Physiology
Insyirah, Samsuddin
Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
description The purification of protein using IMAC can be influenced by imidazole concentration and type of metal ion. Different type of metal ion and different imidazole concentration will give different result of purity and yield for target protein. The strength of binding between histidine- tagged protein and metal ion is varies depending on the type of protein as well as the ion used. Imidazole is one of the competitive agents that are effective at displacing protein. Because of this, imidazole is one of the best agent to be used an eluent. Imidazole at low concentration is commonly use to minimise binding of host cell protein and at somewhat higher concentration, imidazole may also decrease the binding of histidine tagged- protein. This paper presents the effect of imidazole concentration and type of metal ion studies on the purification of recombinant green fluorescent protein (GFP) using an affinity chromatography. Escherichia coli (E.coli) BL21 (DE3) strain carrying the pRSETGFP plasmid encoding the GFP was grown in Luria-Bertani (LB) broth at 300C and 200 rpm for 16 hours. An induction of protein expression, Isopropyl β-ᴅ-1- thiogalactopyranoside when the cell density (OD600) reaches 0.6-0.8 enhanced the yield of functional GFP production. After harvesting, the cell suspension was disrupted using freeze and thaw method. Clarified GFP was then filter using nylon filter before being purified using a HisTrapTM FF 1 ml column (GE healthcare). The column was charged with different metal ion (0.1 M NiSO4 or CuSO4) and the concentration of imidazole at elution buffer was varied (100, 200, 300, 400, 500, 600 mM). For protein analysis, the sample was analysed using Lowry method for total protein determination, while amount of GFP was quantified using gel-based imaging method. This method has resulted in 95% purity at 500 mM imidazole concentration and 93% recovery of GFP at 300 mM imidazole concentration when the column was charged with Ni(II) ion while 89% purity at 500 mM imidazole concentration and 95 % recovery at 400 mM at imidazole concentration of GFP when the column was charged with Cu(II) ion
format Undergraduates Project Papers
author Insyirah, Samsuddin
author_facet Insyirah, Samsuddin
author_sort Insyirah, Samsuddin
title Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
title_short Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
title_full Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
title_fullStr Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
title_full_unstemmed Effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
title_sort effect of imidazole concentration and type of metal ion on the purification of recombinant green fluorescent protein using an affinity chromatography
publishDate 2014
url http://umpir.ump.edu.my/id/eprint/8853/
http://umpir.ump.edu.my/id/eprint/8853/
http://umpir.ump.edu.my/id/eprint/8853/1/CD8547.pdf
first_indexed 2023-09-18T22:06:52Z
last_indexed 2023-09-18T22:06:52Z
_version_ 1777414766816395264

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