combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris
To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fu...
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| Veröffentlicht in: | Yeast (Chichester, England) England), 2011-03, Vol.28 (3), p.237-252 |
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| creator | Nett, Juergen H Stadheim, Terrance A Li, Huijuan Bobrowicz, Piotr Hamilton, Stephen R Davidson, Robert C Choi, Byung-Kwon Mitchell, Teresa Bobrowicz, Beata Rittenhour, Alissa Wildt, Stefan Gerngross, Tillman U |
| description | To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation. Copyright © 2011 John Wiley & Sons, Ltd. |
| doi_str_mv | 10.1002/yea.1835 |
| format | Article |
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Copyright © 2011 John Wiley & Sons, Ltd.</description><identifier>ISSN: 0749-503X</identifier><identifier>ISSN: 1097-0061</identifier><identifier>EISSN: 1097-0061</identifier><identifier>DOI: 10.1002/yea.1835</identifier><identifier>PMID: 21360735</identifier><language>eng</language><publisher>Chichester, UK: John Wiley & Sons, Ltd</publisher><subject>combinatorial genetic libraries ; Endoplasmic Reticulum - enzymology ; Glucosyltransferases - genetics ; Glucosyltransferases - metabolism ; glycosylation ; Golgi Apparatus - enzymology ; Golgi targeting ; Mannosidases - genetics ; Mannosidases - metabolism ; Pichia - enzymology ; Pichia - genetics ; Pichia pastoris ; Protein Engineering ; Protein Sorting Signals - genetics ; Protein Transport ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism</subject><ispartof>Yeast (Chichester, England), 2011-03, Vol.28 (3), p.237-252</ispartof><rights>Copyright © 2011 John Wiley & Sons, Ltd.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4105-9554f7343b0f0dbbd81f5a31f05cee08a858fb02335cb90c49fc37f0920ea5ba3</citedby><cites>FETCH-LOGICAL-c4105-9554f7343b0f0dbbd81f5a31f05cee08a858fb02335cb90c49fc37f0920ea5ba3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fyea.1835$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fyea.1835$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,1427,27901,27902,45550,45551,46384,46808</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21360735$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nett, Juergen H</creatorcontrib><creatorcontrib>Stadheim, Terrance A</creatorcontrib><creatorcontrib>Li, Huijuan</creatorcontrib><creatorcontrib>Bobrowicz, Piotr</creatorcontrib><creatorcontrib>Hamilton, Stephen R</creatorcontrib><creatorcontrib>Davidson, Robert C</creatorcontrib><creatorcontrib>Choi, Byung-Kwon</creatorcontrib><creatorcontrib>Mitchell, Teresa</creatorcontrib><creatorcontrib>Bobrowicz, Beata</creatorcontrib><creatorcontrib>Rittenhour, Alissa</creatorcontrib><creatorcontrib>Wildt, Stefan</creatorcontrib><creatorcontrib>Gerngross, Tillman U</creatorcontrib><title>combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris</title><title>Yeast (Chichester, England)</title><addtitle>Yeast</addtitle><description>To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. 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Copyright © 2011 John Wiley & Sons, Ltd.</description><subject>combinatorial genetic libraries</subject><subject>Endoplasmic Reticulum - enzymology</subject><subject>Glucosyltransferases - genetics</subject><subject>Glucosyltransferases - metabolism</subject><subject>glycosylation</subject><subject>Golgi Apparatus - enzymology</subject><subject>Golgi targeting</subject><subject>Mannosidases - genetics</subject><subject>Mannosidases - metabolism</subject><subject>Pichia - enzymology</subject><subject>Pichia - genetics</subject><subject>Pichia pastoris</subject><subject>Protein Engineering</subject><subject>Protein Sorting Signals - genetics</subject><subject>Protein Transport</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><issn>0749-503X</issn><issn>1097-0061</issn><issn>1097-0061</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1DAQhi0EoktB4gnAN7ikjON44xyrqi1IlYoEleBkTbx21siJg50IhUfhaet0F24VB2tkzadPM_MT8prBGQMoPywGz5jk4gnZMGjqAmDLnpIN1FVTCODfTsiLlH4AMCZK-ZyclIxvoeZiQ_7o0LduwClEh552ZjCT09S7NmJcKI5jDKj3dAp0wtiZie7NZGLwoQtzop1fdEiLx8mFgZrh99Kb9ADvTf7uwugx9VkYV-3s556G-NC8Dr5zqx8jTtkULP3s9N4hHTGt06SX5JlFn8yrYz0ld1eXXy8-Fje3158uzm8KXTEQRSNEZWte8RYs7Np2J5kVyJkFoY0BiVJI20LJudBtA7pqrOa1haYEg6JFfkreHbx51Z-zSZPqXdLGexxM3lFJyaHiIr__kqISW1lLkcn3B1LHkFI0Vo3R9fmiioFaI1M5MrVGltE3R-nc9mb3D_ybUQaKA_DLebM8KlLfL8-PwrcH3mJQ2OVLqrsvJTAOrKmacsv4PUtNrTo</recordid><startdate>201103</startdate><enddate>201103</enddate><creator>Nett, Juergen H</creator><creator>Stadheim, Terrance A</creator><creator>Li, Huijuan</creator><creator>Bobrowicz, Piotr</creator><creator>Hamilton, Stephen R</creator><creator>Davidson, Robert C</creator><creator>Choi, Byung-Kwon</creator><creator>Mitchell, Teresa</creator><creator>Bobrowicz, Beata</creator><creator>Rittenhour, Alissa</creator><creator>Wildt, Stefan</creator><creator>Gerngross, Tillman U</creator><general>John Wiley & Sons, Ltd</general><scope>FBQ</scope><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>7X8</scope><scope>8FD</scope><scope>FR3</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>201103</creationdate><title>combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris</title><author>Nett, Juergen H ; Stadheim, Terrance A ; Li, Huijuan ; Bobrowicz, Piotr ; Hamilton, Stephen R ; Davidson, Robert C ; Choi, Byung-Kwon ; Mitchell, Teresa ; Bobrowicz, Beata ; Rittenhour, Alissa ; Wildt, Stefan ; Gerngross, Tillman U</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4105-9554f7343b0f0dbbd81f5a31f05cee08a858fb02335cb90c49fc37f0920ea5ba3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>combinatorial genetic libraries</topic><topic>Endoplasmic Reticulum - enzymology</topic><topic>Glucosyltransferases - genetics</topic><topic>Glucosyltransferases - metabolism</topic><topic>glycosylation</topic><topic>Golgi Apparatus - enzymology</topic><topic>Golgi targeting</topic><topic>Mannosidases - genetics</topic><topic>Mannosidases - metabolism</topic><topic>Pichia - enzymology</topic><topic>Pichia - genetics</topic><topic>Pichia pastoris</topic><topic>Protein Engineering</topic><topic>Protein Sorting Signals - genetics</topic><topic>Protein Transport</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nett, Juergen H</creatorcontrib><creatorcontrib>Stadheim, Terrance A</creatorcontrib><creatorcontrib>Li, Huijuan</creatorcontrib><creatorcontrib>Bobrowicz, Piotr</creatorcontrib><creatorcontrib>Hamilton, Stephen R</creatorcontrib><creatorcontrib>Davidson, Robert C</creatorcontrib><creatorcontrib>Choi, Byung-Kwon</creatorcontrib><creatorcontrib>Mitchell, Teresa</creatorcontrib><creatorcontrib>Bobrowicz, Beata</creatorcontrib><creatorcontrib>Rittenhour, Alissa</creatorcontrib><creatorcontrib>Wildt, Stefan</creatorcontrib><creatorcontrib>Gerngross, Tillman U</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Yeast (Chichester, England)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nett, Juergen H</au><au>Stadheim, Terrance A</au><au>Li, Huijuan</au><au>Bobrowicz, Piotr</au><au>Hamilton, Stephen R</au><au>Davidson, Robert C</au><au>Choi, Byung-Kwon</au><au>Mitchell, Teresa</au><au>Bobrowicz, Beata</au><au>Rittenhour, Alissa</au><au>Wildt, Stefan</au><au>Gerngross, Tillman U</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris</atitle><jtitle>Yeast (Chichester, England)</jtitle><addtitle>Yeast</addtitle><date>2011-03</date><risdate>2011</risdate><volume>28</volume><issue>3</issue><spage>237</spage><epage>252</epage><pages>237-252</pages><issn>0749-503X</issn><issn>1097-0061</issn><eissn>1097-0061</eissn><abstract>To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. 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| subjects | combinatorial genetic libraries Endoplasmic Reticulum - enzymology Glucosyltransferases - genetics Glucosyltransferases - metabolism glycosylation Golgi Apparatus - enzymology Golgi targeting Mannosidases - genetics Mannosidases - metabolism Pichia - enzymology Pichia - genetics Pichia pastoris Protein Engineering Protein Sorting Signals - genetics Protein Transport Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism |
| title | combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris |
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