Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation
Phospholipase A 2 (PLA 2 ) has found extensive use in industry. However, recombinant PLA 2 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 2 from Strept...
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| Veröffentlicht in: | Applied biochemistry and biotechnology 2021-05, Vol.193 (5), p.1351-1364 |
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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
2
(PLA
2
) has found extensive use in industry. However, recombinant PLA
2
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
2
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
2
protein were similar to those of the native
Streptomyces violaceoruber
PLA
2
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed. |
| doi_str_mv | 10.1007/s12010-020-03333-7 |
| format | Article |
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2
(PLA
2
) has found extensive use in industry. However, recombinant PLA
2
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
2
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
2
protein were similar to those of the native
Streptomyces violaceoruber
PLA
2
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.</description><identifier>ISSN: 0273-2289</identifier><identifier>EISSN: 1559-0291</identifier><identifier>DOI: 10.1007/s12010-020-03333-7</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Biochemistry ; Biotechnology ; Cell culture ; Cell membranes ; Chemistry ; Chemistry and Materials Science ; Enzymes ; Fungi ; Inactivation ; Phospholipase ; Phospholipase A2 ; Proteins ; Splicing ; Streptomyces ; Toxicity ; Yeast ; Yeasts</subject><ispartof>Applied biochemistry and biotechnology, 2021-05, Vol.193 (5), p.1351-1364</ispartof><rights>Springer Science+Business Media, LLC, part of Springer Nature 2020</rights><rights>Springer Science+Business Media, LLC, part of Springer Nature 2020.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3047-7e6d11d8cfa1d138208f924d90af9fb15304d144d33bd67c0224446797abdf943</citedby><cites>FETCH-LOGICAL-c3047-7e6d11d8cfa1d138208f924d90af9fb15304d144d33bd67c0224446797abdf943</cites><orcidid>0000-0003-3236-5103</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s12010-020-03333-7$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s12010-020-03333-7$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids></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 A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation</title><title>Applied biochemistry and biotechnology</title><addtitle>Appl Biochem Biotechnol</addtitle><description>Phospholipase A
2
(PLA
2
) has found extensive use in industry. However, recombinant PLA
2
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
2
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
2
protein were similar to those of the native
Streptomyces violaceoruber
PLA
2
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.</description><subject>Biochemistry</subject><subject>Biotechnology</subject><subject>Cell culture</subject><subject>Cell membranes</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Enzymes</subject><subject>Fungi</subject><subject>Inactivation</subject><subject>Phospholipase</subject><subject>Phospholipase A2</subject><subject>Proteins</subject><subject>Splicing</subject><subject>Streptomyces</subject><subject>Toxicity</subject><subject>Yeast</subject><subject>Yeasts</subject><issn>0273-2289</issn><issn>1559-0291</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>BENPR</sourceid><recordid>eNp9kU9LAzEQxYMoWKtfwFPAi5fVTDa72Rxr_QsVe7AHTyHdZNuU7WZNUrHf3tQKggcDYSDze2_CPITOgVwBIfw6ACVAMkLTzdPJ-AEaQFGI9CTgEA0I5XlGaSWO0UkIK0KAVgUfoHj32XsTgnUddg1-tItlu8WjOtoPg29UHY23qsXTpQv90rW2V8HgEcW2w29GhYhnwXYL_NRFY7vs2WirotH41rRqm-rUu10DjzbRqZ2pimnSKTpqVBvM2U8dotn93ev4MZu8PDyNR5OszgnjGTelBtBV3SjQkFeUVI2gTAuiGtHMoUiUBsZ0ns91yWtCKWOs5IKruW4Ey4focu_be_e-MSHKtQ21aVvVGbcJkjICkDRQJvTiD7pyG9-l30lagCg4KdNah4juqdq7ELxpZO_tWvmtBCJ3Qch9EDIFIb-DkDyJ8r0oJLhbGP9r_Y_qC3anipo</recordid><startdate>20210501</startdate><enddate>20210501</enddate><creator>Cheperegin, S. 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E. ; Malysheva, A. V. ; Sannikova, E. P. ; Gubaidullin, I. I. ; Efremov, B. D. ; Kozlov, D. G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3047-7e6d11d8cfa1d138208f924d90af9fb15304d144d33bd67c0224446797abdf943</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Biochemistry</topic><topic>Biotechnology</topic><topic>Cell culture</topic><topic>Cell membranes</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Enzymes</topic><topic>Fungi</topic><topic>Inactivation</topic><topic>Phospholipase</topic><topic>Phospholipase A2</topic><topic>Proteins</topic><topic>Splicing</topic><topic>Streptomyces</topic><topic>Toxicity</topic><topic>Yeast</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Cheperegin, S. E.</creatorcontrib><creatorcontrib>Malysheva, A. 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E.</au><au>Malysheva, A. V.</au><au>Sannikova, E. P.</au><au>Gubaidullin, I. I.</au><au>Efremov, B. D.</au><au>Kozlov, D. G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation</atitle><jtitle>Applied biochemistry and biotechnology</jtitle><stitle>Appl Biochem Biotechnol</stitle><date>2021-05-01</date><risdate>2021</risdate><volume>193</volume><issue>5</issue><spage>1351</spage><epage>1364</epage><pages>1351-1364</pages><issn>0273-2289</issn><eissn>1559-0291</eissn><abstract>Phospholipase A
2
(PLA
2
) has found extensive use in industry. However, recombinant PLA
2
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
2
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
2
protein were similar to those of the native
Streptomyces violaceoruber
PLA
2
protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.</abstract><cop>New York</cop><pub>Springer US</pub><doi>10.1007/s12010-020-03333-7</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0003-3236-5103</orcidid></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0273-2289 |
| ispartof | Applied biochemistry and biotechnology, 2021-05, Vol.193 (5), p.1351-1364 |
| issn | 0273-2289 1559-0291 |
| language | eng |
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| source | SpringerLink Journals |
| subjects | Biochemistry Biotechnology Cell culture Cell membranes Chemistry Chemistry and Materials Science Enzymes Fungi Inactivation Phospholipase Phospholipase A2 Proteins Splicing Streptomyces Toxicity Yeast Yeasts |
| title | Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation |
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