Expression of Highly Active Bacterial Phospholipase A 2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation
Phospholipase A (PLA ) has found extensive use in industry. However, recombinant PLA production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA from Streptomyces v...
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| Veröffentlicht in: | Applied biochemistry and biotechnology 2021-05, Vol.193 (5), p.1351 |
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| container_title | Applied biochemistry and biotechnology |
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| creator | Cheperegin, S E Malysheva, A V Sannikova, E P Gubaidullin, I I Efremov, B D Kozlov, D G |
| description | Phospholipase A
(PLA
) has found extensive use in industry. However, recombinant PLA
production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA
from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A
protein were similar to those of the native Streptomyces violaceoruber PLA
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed. |
| format | Article |
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(PLA
) has found extensive use in industry. However, recombinant PLA
production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA
from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A
protein were similar to those of the native Streptomyces violaceoruber PLA
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.</description><identifier>EISSN: 1559-0291</identifier><identifier>PMID: 32388605</identifier><language>eng</language><publisher>United States</publisher><ispartof>Applied biochemistry and biotechnology, 2021-05, Vol.193 (5), p.1351</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><orcidid>0000-0003-3236-5103</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32388605$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Cheperegin, S E</creatorcontrib><creatorcontrib>Malysheva, A V</creatorcontrib><creatorcontrib>Sannikova, E P</creatorcontrib><creatorcontrib>Gubaidullin, I I</creatorcontrib><creatorcontrib>Efremov, B D</creatorcontrib><creatorcontrib>Kozlov, D G</creatorcontrib><title>Expression of Highly Active Bacterial Phospholipase A 2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation</title><title>Applied biochemistry and biotechnology</title><addtitle>Appl Biochem Biotechnol</addtitle><description>Phospholipase A
(PLA
) has found extensive use in industry. However, recombinant PLA
production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA
from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A
protein were similar to those of the native Streptomyces violaceoruber PLA
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(PLA
) has found extensive use in industry. However, recombinant PLA
production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA
from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A
protein were similar to those of the native Streptomyces violaceoruber PLA
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.</abstract><cop>United States</cop><pmid>32388605</pmid><orcidid>https://orcid.org/0000-0003-3236-5103</orcidid></addata></record> |
| fulltext | fulltext |
| identifier | EISSN: 1559-0291 |
| ispartof | Applied biochemistry and biotechnology, 2021-05, Vol.193 (5), p.1351 |
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| language | eng |
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| source | SpringerNature Journals |
| title | Expression of Highly Active Bacterial Phospholipase A 2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation |
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