Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase
The intrinsic propensity of each amino acid residue forming α-helices in a protein determines the stability of a protein. The purpose of this study is to improve the stability of the protein by stabilizing the N-terminal α-helix. Therefore, mutations were focused on N-terminal helix of J15 lipase de...
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| Format: | Project Paper Report |
| Language: | English |
| Published: |
2015
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| Online Access: | http://psasir.upm.edu.my/id/eprint/91040/1/FBSB%202015%20150%20-%20IR.pdf |
| _version_ | 1796982436977967104 |
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| author | Lee, Mei Ying |
| author_facet | Lee, Mei Ying |
| author_sort | Lee, Mei Ying |
| collection | UPM |
| description | The intrinsic propensity of each amino acid residue forming α-helices in a protein determines the stability of a protein. The purpose of this study is to improve the stability of the protein by stabilizing the N-terminal α-helix. Therefore, mutations were focused on N-terminal helix of J15 lipase derived from Photobacterium sp. strain J15. The structure of J15 lipase was modeled based on M37 lipase crystal structure as a template. Asp6 of N-terminal helix was chosen as competition of Asp6 with the adjoining main chain hydrogen bonding interaction could destabilize the N-terminal helical structure. In silico analysis using SDM and I-mutant 2.0 revealed that D6L and D6I enhanced the stability of mutated J15 lipases with G of 5.12 kcal/mol and 0.82 kcal/mol respectively. In order to validate the in-silico prediction, the gene encoding J15 lipase was cloned into pEasy-E2 expression vector and transformed into Escherichia coli BL21(De3). Point mutation was generated using Fast Mutagenesis System to increase the mutation efficiency. The Aspartate residue at the sixth position of the N-terminal α-helix was substituted with Isoleucine and Leucine. The wild-type and mutants were induced with 0.015 mM IPTG at 30°C for 16 h. The expected size of the protein was approximately 40.11kDa. Both the wild-type and mutants showed low expression level and most of the proteins were present as insoluble bodies. The specific activity of the crude protein calculated for J15 lipase is 1.953 U/mg, D6I is 1.55 U/mg and D6L is 1.429 U/mg. Overall, the specific activity was low. Due to time constraint, further experimental work could not be performed on time and it was not able to validate the result with the in-silico protein stability predictions. |
| first_indexed | 2024-03-06T10:52:02Z |
| format | Project Paper Report |
| id | upm.eprints-91040 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T10:52:02Z |
| publishDate | 2015 |
| record_format | dspace |
| spelling | upm.eprints-910402021-10-25T04:18:18Z http://psasir.upm.edu.my/id/eprint/91040/ Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase Lee, Mei Ying The intrinsic propensity of each amino acid residue forming α-helices in a protein determines the stability of a protein. The purpose of this study is to improve the stability of the protein by stabilizing the N-terminal α-helix. Therefore, mutations were focused on N-terminal helix of J15 lipase derived from Photobacterium sp. strain J15. The structure of J15 lipase was modeled based on M37 lipase crystal structure as a template. Asp6 of N-terminal helix was chosen as competition of Asp6 with the adjoining main chain hydrogen bonding interaction could destabilize the N-terminal helical structure. In silico analysis using SDM and I-mutant 2.0 revealed that D6L and D6I enhanced the stability of mutated J15 lipases with G of 5.12 kcal/mol and 0.82 kcal/mol respectively. In order to validate the in-silico prediction, the gene encoding J15 lipase was cloned into pEasy-E2 expression vector and transformed into Escherichia coli BL21(De3). Point mutation was generated using Fast Mutagenesis System to increase the mutation efficiency. The Aspartate residue at the sixth position of the N-terminal α-helix was substituted with Isoleucine and Leucine. The wild-type and mutants were induced with 0.015 mM IPTG at 30°C for 16 h. The expected size of the protein was approximately 40.11kDa. Both the wild-type and mutants showed low expression level and most of the proteins were present as insoluble bodies. The specific activity of the crude protein calculated for J15 lipase is 1.953 U/mg, D6I is 1.55 U/mg and D6L is 1.429 U/mg. Overall, the specific activity was low. Due to time constraint, further experimental work could not be performed on time and it was not able to validate the result with the in-silico protein stability predictions. 2015-06 Project Paper Report NonPeerReviewed text en http://psasir.upm.edu.my/id/eprint/91040/1/FBSB%202015%20150%20-%20IR.pdf Lee, Mei Ying (2015) Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase. [Project Paper Report] |
| spellingShingle | Lee, Mei Ying Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase |
| title | Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase |
| title_full | Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase |
| title_fullStr | Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase |
| title_full_unstemmed | Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase |
| title_short | Site-directed mutagenesis of n-terminal Alpha Helix of J15 lipase |
| title_sort | site directed mutagenesis of n terminal alpha helix of j15 lipase |
| url | http://psasir.upm.edu.my/id/eprint/91040/1/FBSB%202015%20150%20-%20IR.pdf |
| work_keys_str_mv | AT leemeiying sitedirectedmutagenesisofnterminalalphahelixofj15lipase |