Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease.
Polyprotein processing of dengue virus is performed by the host signal peptidase and virus encoded two-component protease consisting of NS3 and its cofactor NS2B. The exact mechanism by which NS2B cofactor stimulates the NS3 protease is not known and structure of complete NS2B is still lacking. To s...
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| Format: | Final Year Project (FYP) |
| Language: | English |
| Published: |
2009
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| Online Access: | http://hdl.handle.net/10356/16305 |
| _version_ | 1811696813887979520 |
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| author | Kwek, Stanley Ming Kun. |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Kwek, Stanley Ming Kun. |
| author_sort | Kwek, Stanley Ming Kun. |
| collection | NTU |
| description | Polyprotein processing of dengue virus is performed by the host signal peptidase and virus encoded two-component protease consisting of NS3 and its cofactor NS2B. The exact mechanism by which NS2B cofactor stimulates the NS3 protease is not known and structure of complete NS2B is still lacking. To study the role of NS2B cofactor in the protease activity, we expressed in Escherichia coil the full length NS2B integral membrane protein together with dengue serotype 2 NS3 of various truncations, namely, NS2B-NS371aa, NS2B-NS3Pro and NS2B-NS3FL. The expression of functional and soluble NS2B-NS3Pro and NS2B-NS3FL proteins, showed high level of protease activity as auto-proteolysis at several sites of the protein were observed. NS2B-NS371aa which yielded an intact protein band was chosen for protein purification for the purpose of obtaining intact and pure NS2B with truncated NS3. The highest expression level of NS2B-NS371aa was by the auto-induction expression. Detergent solubilization with n-dodecyl-β-d-maltoside was effective that extracted the largest quantity of soluble NS2B-NS371aa. Ni-NTA affinity chromatography together with cation exchange chromatography proved useful in removing most Escherichia coil proteins during purification but the low expression level associated with NS2B-NS371aa remains a major hurdle. This study serves as a foundation and forms the preliminary work towards hydrophobic studies on full length NS2B integral membrane protein. |
| first_indexed | 2024-10-01T07:45:20Z |
| format | Final Year Project (FYP) |
| id | ntu-10356/16305 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T07:45:20Z |
| publishDate | 2009 |
| record_format | dspace |
| spelling | ntu-10356/163052023-02-28T18:02:10Z Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. Kwek, Stanley Ming Kun. School of Biological Sciences Duke-NUS Medical School Subhash Vasudevan DRNTU::Science::Biological sciences::Molecular biology Polyprotein processing of dengue virus is performed by the host signal peptidase and virus encoded two-component protease consisting of NS3 and its cofactor NS2B. The exact mechanism by which NS2B cofactor stimulates the NS3 protease is not known and structure of complete NS2B is still lacking. To study the role of NS2B cofactor in the protease activity, we expressed in Escherichia coil the full length NS2B integral membrane protein together with dengue serotype 2 NS3 of various truncations, namely, NS2B-NS371aa, NS2B-NS3Pro and NS2B-NS3FL. The expression of functional and soluble NS2B-NS3Pro and NS2B-NS3FL proteins, showed high level of protease activity as auto-proteolysis at several sites of the protein were observed. NS2B-NS371aa which yielded an intact protein band was chosen for protein purification for the purpose of obtaining intact and pure NS2B with truncated NS3. The highest expression level of NS2B-NS371aa was by the auto-induction expression. Detergent solubilization with n-dodecyl-β-d-maltoside was effective that extracted the largest quantity of soluble NS2B-NS371aa. Ni-NTA affinity chromatography together with cation exchange chromatography proved useful in removing most Escherichia coil proteins during purification but the low expression level associated with NS2B-NS371aa remains a major hurdle. This study serves as a foundation and forms the preliminary work towards hydrophobic studies on full length NS2B integral membrane protein. Bachelor of Science in Biological Sciences 2009-05-25T03:38:14Z 2009-05-25T03:38:14Z 2009 2009 Final Year Project (FYP) http://hdl.handle.net/10356/16305 en Nanyang Technological University 44 p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences::Molecular biology Kwek, Stanley Ming Kun. Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. |
| title | Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. |
| title_full | Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. |
| title_fullStr | Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. |
| title_full_unstemmed | Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. |
| title_short | Expression of the full length NS2B integral membrane protein, a cofactor for dengue protease. |
| title_sort | expression of the full length ns2b integral membrane protein a cofactor for dengue protease |
| topic | DRNTU::Science::Biological sciences::Molecular biology |
| url | http://hdl.handle.net/10356/16305 |
| work_keys_str_mv | AT kwekstanleymingkun expressionofthefulllengthns2bintegralmembraneproteinacofactorfordengueprotease |