Expression, purification, and oligomerization of phosphoprotein of nipah virus

Nipah virus (NiV) is a single-stranded, non-segmented negative-sense RNA virus belonging to genus Henipavirus. NiV causes fatality to humans and various types of animals. Its genome encodes six major structural proteins: nucleocapsid (N), phospho (P), matrix (M), fusion (F), glyco (G) and large (L)...

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Main Author:	Salvamani, Shamala
Format:	Thesis
Language:	English
Published:	2013
Subjects:	Nipah virus Phosphoproteins Oligomerization
Online Access:	http://psasir.upm.edu.my/id/eprint/42831/1/FBSB%202013%2029R.pdf
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spelling	my-upm-ir.428312016-06-24T04:22:35Z Expression, purification, and oligomerization of phosphoprotein of nipah virus 2013-05 Salvamani, Shamala Nipah virus (NiV) is a single-stranded, non-segmented negative-sense RNA virus belonging to genus Henipavirus. NiV causes fatality to humans and various types of animals. Its genome encodes six major structural proteins: nucleocapsid (N), phospho (P), matrix (M), fusion (F), glyco (G) and large (L) proteins. The P protein is vital for the genome replication and transcription. The potential diagnostic utility of the purified recombinant NiV P protein was determined in this study. In addition, the oligomeric nature of the P protein was also explored through its multimerization domain. Previously, the NiV P gene (2124 bp) was cloned into pTrcHis2-TOPO vector. In the present study, the P protein (120 kDa) was expressed in Escherichia coli and the optimal expression conditions such as the cultivation temperature, concentration of IPTG and the time of post-induction were determined in order to express the P protein in soluble form. SDS-PAGE and Western blot analysis using the anti-His antibody confirmed the protein expression. The optimum cultivation temperature for the recombinant protein expression was at 37 ºC while the optimum induction time was at 9 h with 1 mM IPTG. Solubility analysis showed that about 70% of the P protein was in soluble form at 37 ºC. An immobilized metal affinity chromatography (IMAC) was used to purify the recombinant P protein from the clarified E. coli homogenate. The purity of elution after HisTrap purification was 92.67% with a purification factor of 11.58. ELISA and Western blot analysis confirmed that the P protein was antigenic and could be used in serodiagnosis for detecting anti-P antibody of NiV infections. The oligomerization of P protein plays essential roles in the transcription and replication cycle of NiV. The full length P protein has different phosphorylation status which leads to the existence of more than one protein band in SDS-PAGE and Western blot, it is very difficult to determine the oligomeric nature of the P protein. Thus, the phosphoprotein multimerization domain (PMD) of NiV, which is responsible for the formation of oligomers, was cloned into pTrcHis2-TOPO vector, expressed in E. coli and purified with His-Trap column. A stepwise elution protocol was used in order to obtain purer recombinant PMD protein which yielded about 96.73% purity. The purified PMD protein was subjected to chemical cross-linking and dynamic light scattering analyses. The result confirmed that the PMD protein of NiV form tetramers. Circular dichroism analysis revealed that the PMD domain has a high α-helical content, about 55%. This study demonstrated the potential of NiV P protein as a diagnostic reagent for the detection of NiV infections and the oligomeric state of its multimerization domain. Nipah virus Phosphoproteins Oligomerization 2013-05 Thesis http://psasir.upm.edu.my/id/eprint/42831/ http://psasir.upm.edu.my/id/eprint/42831/1/FBSB%202013%2029R.pdf application/pdf en public masters Universiti Putra Malaysia Nipah virus Phosphoproteins Oligomerization
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Nipah virus Phosphoproteins Oligomerization
spellingShingle	Nipah virus Phosphoproteins Oligomerization Salvamani, Shamala Expression, purification, and oligomerization of phosphoprotein of nipah virus
description	Nipah virus (NiV) is a single-stranded, non-segmented negative-sense RNA virus belonging to genus Henipavirus. NiV causes fatality to humans and various types of animals. Its genome encodes six major structural proteins: nucleocapsid (N), phospho (P), matrix (M), fusion (F), glyco (G) and large (L) proteins. The P protein is vital for the genome replication and transcription. The potential diagnostic utility of the purified recombinant NiV P protein was determined in this study. In addition, the oligomeric nature of the P protein was also explored through its multimerization domain. Previously, the NiV P gene (2124 bp) was cloned into pTrcHis2-TOPO vector. In the present study, the P protein (120 kDa) was expressed in Escherichia coli and the optimal expression conditions such as the cultivation temperature, concentration of IPTG and the time of post-induction were determined in order to express the P protein in soluble form. SDS-PAGE and Western blot analysis using the anti-His antibody confirmed the protein expression. The optimum cultivation temperature for the recombinant protein expression was at 37 ºC while the optimum induction time was at 9 h with 1 mM IPTG. Solubility analysis showed that about 70% of the P protein was in soluble form at 37 ºC. An immobilized metal affinity chromatography (IMAC) was used to purify the recombinant P protein from the clarified E. coli homogenate. The purity of elution after HisTrap purification was 92.67% with a purification factor of 11.58. ELISA and Western blot analysis confirmed that the P protein was antigenic and could be used in serodiagnosis for detecting anti-P antibody of NiV infections. The oligomerization of P protein plays essential roles in the transcription and replication cycle of NiV. The full length P protein has different phosphorylation status which leads to the existence of more than one protein band in SDS-PAGE and Western blot, it is very difficult to determine the oligomeric nature of the P protein. Thus, the phosphoprotein multimerization domain (PMD) of NiV, which is responsible for the formation of oligomers, was cloned into pTrcHis2-TOPO vector, expressed in E. coli and purified with His-Trap column. A stepwise elution protocol was used in order to obtain purer recombinant PMD protein which yielded about 96.73% purity. The purified PMD protein was subjected to chemical cross-linking and dynamic light scattering analyses. The result confirmed that the PMD protein of NiV form tetramers. Circular dichroism analysis revealed that the PMD domain has a high α-helical content, about 55%. This study demonstrated the potential of NiV P protein as a diagnostic reagent for the detection of NiV infections and the oligomeric state of its multimerization domain.
format	Thesis
qualification_level	Master's degree
author	Salvamani, Shamala
author_facet	Salvamani, Shamala
author_sort	Salvamani, Shamala
title	Expression, purification, and oligomerization of phosphoprotein of nipah virus
title_short	Expression, purification, and oligomerization of phosphoprotein of nipah virus
title_full	Expression, purification, and oligomerization of phosphoprotein of nipah virus
title_fullStr	Expression, purification, and oligomerization of phosphoprotein of nipah virus
title_full_unstemmed	Expression, purification, and oligomerization of phosphoprotein of nipah virus
title_sort	expression, purification, and oligomerization of phosphoprotein of nipah virus
granting_institution	Universiti Putra Malaysia
publishDate	2013
url	http://psasir.upm.edu.my/id/eprint/42831/1/FBSB%202013%2029R.pdf
_version_	1747811914062233600

Expression, purification, and oligomerization of phosphoprotein of nipah virus

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