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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| Format: | Thesis |
| Language: | English |
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2013
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| Online Access: | http://psasir.upm.edu.my/id/eprint/42831/1/FBSB%202013%2029R.pdf |
| _version_ | 1796974224470966272 |
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| author | Salvamani, Shamala |
| author_facet | Salvamani, Shamala |
| author_sort | Salvamani, Shamala |
| collection | UPM |
| 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. |
| first_indexed | 2024-03-06T08:53:57Z |
| format | Thesis |
| id | upm.eprints-42831 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T08:53:57Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | upm.eprints-428312016-06-24T04:22:35Z http://psasir.upm.edu.my/id/eprint/42831/ Expression, purification, and oligomerization of phosphoprotein of nipah virus 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. 2013-05 Thesis NonPeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/42831/1/FBSB%202013%2029R.pdf Salvamani, Shamala (2013) Expression, purification, and oligomerization of phosphoprotein of nipah virus. Masters thesis, Universiti Putra Malaysia. Nipah virus Phosphoproteins Oligomerization |
| spellingShingle | Nipah virus Phosphoproteins Oligomerization Salvamani, Shamala Expression, purification, and oligomerization of phosphoprotein of nipah virus |
| title | 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_short | Expression, purification, and oligomerization of phosphoprotein of nipah virus |
| title_sort | expression purification and oligomerization of phosphoprotein of nipah virus |
| topic | Nipah virus Phosphoproteins Oligomerization |
| url | http://psasir.upm.edu.my/id/eprint/42831/1/FBSB%202013%2029R.pdf |
| work_keys_str_mv | AT salvamanishamala expressionpurificationandoligomerizationofphosphoproteinofnipahvirus |