Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda

Insect cell-baculovirus system is an excellent artificial system for the production of recombinant glycoprotein despite its glycosylation deficiencies. In this study, laboratory scale production of recombinant human transferrin (rhTf) from insect cell-BEVS was conducted and chromatographic purificat...

Full description

Saved in:

Bibliographic Details
Main Author:	Wee, Chen Chen
Format:	Thesis
Language:	English
Published:	2008
Subjects:	TP Chemical technology QH301 Biology
Online Access:	http://eprints.utm.my/id/eprint/9647/1/WeeChenChenMFKT2008.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-utm-ep.9647
record_format	uketd_dc
spelling	my-utm-ep.96472018-10-14T07:19:59Z Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda 2008-06 Wee, Chen Chen TP Chemical technology QH301 Biology Insect cell-baculovirus system is an excellent artificial system for the production of recombinant glycoprotein despite its glycosylation deficiencies. In this study, laboratory scale production of recombinant human transferrin (rhTf) from insect cell-BEVS was conducted and chromatographic purification strategies were employed to obtain rhTf in high yield and high recovery. Research was started with the amplification of recombinant baculovirus, using low multiplicity of infection (MOI). Virus stock in a 1.2 x 109 pfu/ml infected suspension culture of Spodoptera frugiperda (Sf9) at 15 MOI had produced 31Âµg/ml of rhTf. To purify the rhTf, hydrophobic interaction chromatography, dialysis and ion exchange chromatography were performed. For hydrophobic interaction chromatography, elution strategy, flowrate and rhTf loading capacity of phenyl sepharose were optimized. By loading 38Âµg rhTf/ml of gel, employing step elution with 50% 1.2M (NH4)2SO4/0.4M Na3C6H5O7, pH6 (buffer A) and 25% buffer A and flowrate at 1ml/min, 74.6% of rhTf had been recovered from phenyl sepharose. For ion exchange chromatography, batch purification in reduced size was used to select suitable anion exchange matrix, suitable pH of equilibration buffer and concentration of equilibration buffer. 20mM Tris/HCl buffer, pH8.5 and gradient elution with the increase of of 5mM NaCl/CV succeeded in giving pure rhTf with 52.5% recovery from Q-sepharose. The overall recovery of pure rhTf was 34% with 200 purification fold. A brief glycan characterization of the recovered pure rhTf was performed for a better understanding of the glycosylation feature of this protein expressed using optimized medium from BEVS. The carbohydrate component of the purified rhTf was determined. The purified rhTf was hydrolyzed and the release sugar was labeled with 1-Phenyl-3- Methyl-5-Pyrazolone (PMP) before analysis with High performance Liquid Chromatography (HPLC). The molar fractions of Man, GlcNAc and Gal of rhTf were 3.78, 1.69 and 0.93, respectively. 2008-06 Thesis http://eprints.utm.my/id/eprint/9647/ http://eprints.utm.my/id/eprint/9647/1/WeeChenChenMFKT2008.pdf application/pdf en public masters Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering Faculty of Chemical and Natural Resources Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology QH301 Biology
spellingShingle	TP Chemical technology QH301 Biology Wee, Chen Chen Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
description	Insect cell-baculovirus system is an excellent artificial system for the production of recombinant glycoprotein despite its glycosylation deficiencies. In this study, laboratory scale production of recombinant human transferrin (rhTf) from insect cell-BEVS was conducted and chromatographic purification strategies were employed to obtain rhTf in high yield and high recovery. Research was started with the amplification of recombinant baculovirus, using low multiplicity of infection (MOI). Virus stock in a 1.2 x 109 pfu/ml infected suspension culture of Spodoptera frugiperda (Sf9) at 15 MOI had produced 31Âµg/ml of rhTf. To purify the rhTf, hydrophobic interaction chromatography, dialysis and ion exchange chromatography were performed. For hydrophobic interaction chromatography, elution strategy, flowrate and rhTf loading capacity of phenyl sepharose were optimized. By loading 38Âµg rhTf/ml of gel, employing step elution with 50% 1.2M (NH4)2SO4/0.4M Na3C6H5O7, pH6 (buffer A) and 25% buffer A and flowrate at 1ml/min, 74.6% of rhTf had been recovered from phenyl sepharose. For ion exchange chromatography, batch purification in reduced size was used to select suitable anion exchange matrix, suitable pH of equilibration buffer and concentration of equilibration buffer. 20mM Tris/HCl buffer, pH8.5 and gradient elution with the increase of of 5mM NaCl/CV succeeded in giving pure rhTf with 52.5% recovery from Q-sepharose. The overall recovery of pure rhTf was 34% with 200 purification fold. A brief glycan characterization of the recovered pure rhTf was performed for a better understanding of the glycosylation feature of this protein expressed using optimized medium from BEVS. The carbohydrate component of the purified rhTf was determined. The purified rhTf was hydrolyzed and the release sugar was labeled with 1-Phenyl-3- Methyl-5-Pyrazolone (PMP) before analysis with High performance Liquid Chromatography (HPLC). The molar fractions of Man, GlcNAc and Gal of rhTf were 3.78, 1.69 and 0.93, respectively.
format	Thesis
qualification_level	Master's degree
author	Wee, Chen Chen
author_facet	Wee, Chen Chen
author_sort	Wee, Chen Chen
title	Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
title_short	Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
title_full	Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
title_fullStr	Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
title_full_unstemmed	Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
title_sort	chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda
granting_institution	Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering
granting_department	Faculty of Chemical and Natural Resources Engineering
publishDate	2008
url	http://eprints.utm.my/id/eprint/9647/1/WeeChenChenMFKT2008.pdf
_version_	1747814768800956416

Chromatographic purification strategies for recombinant human transferrin from spodoptera frugiperda

Similar Items