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Enhancement of a protocol purifying T1 lipase through molecular approach
T1 Lipase is a thermostable secretary protein of strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhanc...
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| Published in: | PeerJ (San Francisco, CA) CA), 2018-11, Vol.6, p.e5833-e5833, Article e5833 |
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| cites | cdi_FETCH-LOGICAL-c570t-c919b0c6a5eec3f6320ba1c72a8907dce982376db01b3d86f517e0b7f7922e883 |
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| creator | Che Hussian, Che Haznie Ayu Raja Abd Rahman, Raja Noor Zaliha Thean Chor, Adam Leow Salleh, Abu Bakar Mohamad Ali, Mohd Shukuri |
| description | T1 Lipase is a thermostable secretary protein of
strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification. |
| doi_str_mv | 10.7717/peerj.5833 |
| format | article |
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strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.</description><identifier>ISSN: 2167-8359</identifier><identifier>EISSN: 2167-8359</identifier><identifier>DOI: 10.7717/peerj.5833</identifier><identifier>PMID: 30479887</identifier><language>eng</language><publisher>United States: PeerJ. Ltd</publisher><subject>Affinity ; Analysis ; Biocatalysts ; Biochemistry ; Biotechnology ; Chromatography ; Commercialization ; Computational Biology ; E coli ; Enzymes ; Fatty acids ; Gene mutations ; Genetic aspects ; Glutathione transferase ; Isoelectric point ; Lipase ; Lipids ; Microbiology ; Molecular biology ; Mutagenesis ; Mutation ; Protein expression ; Proteins ; Purifications ; Site-directed mutagenesis ; Triglycerides</subject><ispartof>PeerJ (San Francisco, CA), 2018-11, Vol.6, p.e5833-e5833, Article e5833</ispartof><rights>COPYRIGHT 2018 PeerJ. Ltd.</rights><rights>2018 Che Hussian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2018 Che Hussian et al. 2018 Che Hussian et al.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c570t-c919b0c6a5eec3f6320ba1c72a8907dce982376db01b3d86f517e0b7f7922e883</citedby><cites>FETCH-LOGICAL-c570t-c919b0c6a5eec3f6320ba1c72a8907dce982376db01b3d86f517e0b7f7922e883</cites><orcidid>0000-0002-0280-7735</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/2134162633/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/2134162633?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25752,27923,27924,37011,37012,44589,53790,53792,74997</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30479887$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Che Hussian, Che Haznie Ayu</creatorcontrib><creatorcontrib>Raja Abd Rahman, Raja Noor Zaliha</creatorcontrib><creatorcontrib>Thean Chor, Adam Leow</creatorcontrib><creatorcontrib>Salleh, Abu Bakar</creatorcontrib><creatorcontrib>Mohamad Ali, Mohd Shukuri</creatorcontrib><title>Enhancement of a protocol purifying T1 lipase through molecular approach</title><title>PeerJ (San Francisco, CA)</title><addtitle>PeerJ</addtitle><description>T1 Lipase is a thermostable secretary protein of
strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.</description><subject>Affinity</subject><subject>Analysis</subject><subject>Biocatalysts</subject><subject>Biochemistry</subject><subject>Biotechnology</subject><subject>Chromatography</subject><subject>Commercialization</subject><subject>Computational Biology</subject><subject>E coli</subject><subject>Enzymes</subject><subject>Fatty acids</subject><subject>Gene mutations</subject><subject>Genetic aspects</subject><subject>Glutathione transferase</subject><subject>Isoelectric point</subject><subject>Lipase</subject><subject>Lipids</subject><subject>Microbiology</subject><subject>Molecular biology</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Protein expression</subject><subject>Proteins</subject><subject>Purifications</subject><subject>Site-directed mutagenesis</subject><subject>Triglycerides</subject><issn>2167-8359</issn><issn>2167-8359</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNptkl1rFDEUhgdRbKm98QfIgCAibM3HTD5uhFKqLRS8qdchkznZyZJJxmRG6L830611V0wuEpL3PCd5eavqLUYXnGP-eQJIu4tWUPqiOiWY8Y2grXx5sD-pznPeoTIEYUjQ19UJRQ2XQvDT6uY6DDoYGCHMdbS1rqcU52iir6clOfvgwra-x7V3k85Qz0OKy3aox-jBLF6nWk-lQJvhTfXKap_h_Gk9q358vb6_utncff92e3V5tzEtR_PGSCw7ZJhuAQy1jBLUaWw40UIi3huQglDO-g7hjvaC2RZzQB23XBICQtCz6nbP7aPeqSm5UacHFbVTjwcxbZVOszMeFFApC0Z2DbUNQaw01j1uO9MhWhywhfVlz5qWboTSPMxJ-yPo8U1wg9rGX4qRBlPZFsDHJ0CKPxfIsxpdNuC9DhCXrAimglHRsPXd7_-R7uKSQrFqVTWYEUbpX9VWlw-4YGPpa1aoumwZWVFyZV38R1VmD6MzMYB15fyo4MNBwQDaz0OOfpldDPlY-GkvNCnmnMA-m4GRWvOmHvOm1rwV8btD-56lf9JFfwOG085w</recordid><startdate>20181116</startdate><enddate>20181116</enddate><creator>Che Hussian, Che Haznie Ayu</creator><creator>Raja Abd Rahman, Raja Noor Zaliha</creator><creator>Thean Chor, Adam Leow</creator><creator>Salleh, Abu Bakar</creator><creator>Mohamad Ali, Mohd Shukuri</creator><general>PeerJ. 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strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.</abstract><cop>United States</cop><pub>PeerJ. Ltd</pub><pmid>30479887</pmid><doi>10.7717/peerj.5833</doi><orcidid>https://orcid.org/0000-0002-0280-7735</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Affinity Analysis Biocatalysts Biochemistry Biotechnology Chromatography Commercialization Computational Biology E coli Enzymes Fatty acids Gene mutations Genetic aspects Glutathione transferase Isoelectric point Lipase Lipids Microbiology Molecular biology Mutagenesis Mutation Protein expression Proteins Purifications Site-directed mutagenesis Triglycerides |
| title | Enhancement of a protocol purifying T1 lipase through molecular approach |
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