Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy
Although Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pa...
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| Veröffentlicht in: | Microbial cell factories 2015-08, Vol.14 (1), p.123-123, Article 123 |
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| creator | Wang, Ping Huang, Lu Jiang, Hu Tian, Jian Chu, Xiaoyu Wu, Ningfeng |
| description | Although Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris.
The results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.
These results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features. |
| doi_str_mv | 10.1186/s12934-015-0315-4 |
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The results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.
These results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.</description><identifier>ISSN: 1475-2859</identifier><identifier>EISSN: 1475-2859</identifier><identifier>DOI: 10.1186/s12934-015-0315-4</identifier><identifier>PMID: 26310666</identifier><language>eng</language><publisher>England: BioMed Central Ltd</publisher><subject>Amino acids ; Analysis ; Enzymes ; Gene Dosage ; Genetic Engineering ; Health aspects ; Hydrolases ; Organisms, Genetically Modified ; Phosphoric Monoester Hydrolases - metabolism ; Physiological aspects ; Pichia - genetics ; Pichia - metabolism ; Protein C ; Protein Sorting Signals ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; RNA, Messenger - metabolism ; Sequence Analysis, Protein</subject><ispartof>Microbial cell factories, 2015-08, Vol.14 (1), p.123-123, Article 123</ispartof><rights>COPYRIGHT 2015 BioMed Central Ltd.</rights><rights>Copyright BioMed Central 2015</rights><rights>Wang et al. 2015</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c528t-17b49c7256acb9955d457b9b5232ad45bd1c89cd218997f8005914a9984241723</citedby><cites>FETCH-LOGICAL-c528t-17b49c7256acb9955d457b9b5232ad45bd1c89cd218997f8005914a9984241723</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551668/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551668/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27923,27924,53790,53792</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26310666$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wang, Ping</creatorcontrib><creatorcontrib>Huang, Lu</creatorcontrib><creatorcontrib>Jiang, Hu</creatorcontrib><creatorcontrib>Tian, Jian</creatorcontrib><creatorcontrib>Chu, Xiaoyu</creatorcontrib><creatorcontrib>Wu, Ningfeng</creatorcontrib><title>Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy</title><title>Microbial cell factories</title><addtitle>Microb Cell Fact</addtitle><description>Although Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris.
The results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.
These results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.</description><subject>Amino acids</subject><subject>Analysis</subject><subject>Enzymes</subject><subject>Gene Dosage</subject><subject>Genetic Engineering</subject><subject>Health aspects</subject><subject>Hydrolases</subject><subject>Organisms, Genetically Modified</subject><subject>Phosphoric Monoester Hydrolases - metabolism</subject><subject>Physiological aspects</subject><subject>Pichia - genetics</subject><subject>Pichia - metabolism</subject><subject>Protein C</subject><subject>Protein Sorting Signals</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>RNA, Messenger - metabolism</subject><subject>Sequence Analysis, Protein</subject><issn>1475-2859</issn><issn>1475-2859</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNptkklr3TAUhU1paYb2B3RTDN00Cye6ssZNIYSkDQQaOqyFLMu2gi29Snbo-_eReWmaV4pAukjfOZpOUbwDdAog2FkCLGtSIaAVqnNHXhSHQDitsKDy5bP6oDhK6Q4h4ILXr4sDzGpAjLHDor30g_bGtmWyJtrZBV-GrtTlZOdhO5YbHfU8rLPDto1h1MmWzpe3zgxO59U0h-hSuSTn-6wyYWqc16uNHss0Z7Htt2-KV50ek337OB4XP68uf1x8qW6-fr6-OL-pDMViroA3RBqOKdOmkZLSllDeyIbiGutcNy0YIU2LQUjJO4EQlUC0lIJgAhzXx8Wnne9maSbbGuvzAUa1iW7ScauCdmp_xbtB9eFeEUqBMZENPj4axPBrsWlWk0vGjqP2NixJAUdCSAqcZfTDP-hdWGK-9UoJxAihgvylej1a5XwX8r5mNVXnlOT_AYpX6vQ_VG6tnZwJ3nYuz-8JTvYEmZnt77nXS0rq-vu3fRZ2rIkhpWi7p_cApNYYqV2MVI6RWmOkVs375w_5pPiTm_oBF5TA9g</recordid><startdate>20150828</startdate><enddate>20150828</enddate><creator>Wang, Ping</creator><creator>Huang, Lu</creator><creator>Jiang, Hu</creator><creator>Tian, Jian</creator><creator>Chu, Xiaoyu</creator><creator>Wu, Ningfeng</creator><general>BioMed Central Ltd</general><general>BioMed Central</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20150828</creationdate><title>Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy</title><author>Wang, Ping ; Huang, Lu ; Jiang, Hu ; Tian, Jian ; Chu, Xiaoyu ; Wu, Ningfeng</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c528t-17b49c7256acb9955d457b9b5232ad45bd1c89cd218997f8005914a9984241723</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amino acids</topic><topic>Analysis</topic><topic>Enzymes</topic><topic>Gene Dosage</topic><topic>Genetic Engineering</topic><topic>Health aspects</topic><topic>Hydrolases</topic><topic>Organisms, Genetically Modified</topic><topic>Phosphoric Monoester Hydrolases - metabolism</topic><topic>Physiological aspects</topic><topic>Pichia - genetics</topic><topic>Pichia - metabolism</topic><topic>Protein C</topic><topic>Protein Sorting Signals</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>RNA, Messenger - metabolism</topic><topic>Sequence Analysis, Protein</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Ping</creatorcontrib><creatorcontrib>Huang, Lu</creatorcontrib><creatorcontrib>Jiang, Hu</creatorcontrib><creatorcontrib>Tian, Jian</creatorcontrib><creatorcontrib>Chu, Xiaoyu</creatorcontrib><creatorcontrib>Wu, Ningfeng</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Microbial cell factories</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Ping</au><au>Huang, Lu</au><au>Jiang, Hu</au><au>Tian, Jian</au><au>Chu, Xiaoyu</au><au>Wu, Ningfeng</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy</atitle><jtitle>Microbial cell factories</jtitle><addtitle>Microb Cell Fact</addtitle><date>2015-08-28</date><risdate>2015</risdate><volume>14</volume><issue>1</issue><spage>123</spage><epage>123</epage><pages>123-123</pages><artnum>123</artnum><issn>1475-2859</issn><eissn>1475-2859</eissn><abstract>Although Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris.
The results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.
These results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.</abstract><cop>England</cop><pub>BioMed Central Ltd</pub><pmid>26310666</pmid><doi>10.1186/s12934-015-0315-4</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino acids Analysis Enzymes Gene Dosage Genetic Engineering Health aspects Hydrolases Organisms, Genetically Modified Phosphoric Monoester Hydrolases - metabolism Physiological aspects Pichia - genetics Pichia - metabolism Protein C Protein Sorting Signals Recombinant Proteins - genetics Recombinant Proteins - metabolism RNA, Messenger - metabolism Sequence Analysis, Protein |
| title | Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy |
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