Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins
We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations th...
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| Veröffentlicht in: | Journal of structural biology 2016-04, Vol.194 (1), p.1-7 |
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| container_title | Journal of structural biology |
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| creator | Bokhove, Marcel Sadat Al Hosseini, Hamed Saito, Takako Dioguardi, Elisa Gegenschatz-Schmid, Katharina Nishimura, Kaoru Raj, Isha de Sanctis, Daniele Han, Ling Jovine, Luca |
| description | We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. |
| doi_str_mv | 10.1016/j.jsb.2016.01.016 |
| format | Article |
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This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.</description><identifier>ISSN: 1047-8477</identifier><identifier>ISSN: 1095-8657</identifier><identifier>EISSN: 1095-8657</identifier><identifier>DOI: 10.1016/j.jsb.2016.01.016</identifier><identifier>PMID: 26850170</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Animals ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Base Sequence ; CHO Cells ; Cricetinae ; Cricetulus ; Crystallization ; Crystallography, X-Ray ; Gene Expression ; Glycoproteins ; HEK293 Cells ; Humans ; Maltose-binding protein fusion ; Maltose-Binding Proteins - chemistry ; Maltose-Binding Proteins - genetics ; Maltose-Binding Proteins - metabolism ; Mammalian cell expression ; Medicin och hälsovetenskap ; Models, Molecular ; Molecular replacement ; Mutation ; Protein Conformation ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Sf9 Cells ; Technical Note ; X-ray crystallography</subject><ispartof>Journal of structural biology, 2016-04, Vol.194 (1), p.1-7</ispartof><rights>2016 The Authors</rights><rights>Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.</rights><rights>2016 The Authors 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c605t-c6da0247e7a8b785022e383357a341437b99917eef1443cc96a4917a651640d93</citedby><cites>FETCH-LOGICAL-c605t-c6da0247e7a8b785022e383357a341437b99917eef1443cc96a4917a651640d93</cites><orcidid>0000-0002-2679-6946 ; 0000-0001-7241-5967</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jsb.2016.01.016$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,315,554,782,786,887,3554,27933,27934,46004</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26850170$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:133096834$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Bokhove, Marcel</creatorcontrib><creatorcontrib>Sadat Al Hosseini, Hamed</creatorcontrib><creatorcontrib>Saito, Takako</creatorcontrib><creatorcontrib>Dioguardi, Elisa</creatorcontrib><creatorcontrib>Gegenschatz-Schmid, Katharina</creatorcontrib><creatorcontrib>Nishimura, Kaoru</creatorcontrib><creatorcontrib>Raj, Isha</creatorcontrib><creatorcontrib>de Sanctis, Daniele</creatorcontrib><creatorcontrib>Han, Ling</creatorcontrib><creatorcontrib>Jovine, Luca</creatorcontrib><title>Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins</title><title>Journal of structural biology</title><addtitle>J Struct Biol</addtitle><description>We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Base Sequence</subject><subject>CHO Cells</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Crystallization</subject><subject>Crystallography, X-Ray</subject><subject>Gene Expression</subject><subject>Glycoproteins</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Maltose-binding protein fusion</subject><subject>Maltose-Binding Proteins - chemistry</subject><subject>Maltose-Binding Proteins - genetics</subject><subject>Maltose-Binding Proteins - metabolism</subject><subject>Mammalian cell expression</subject><subject>Medicin och hälsovetenskap</subject><subject>Models, Molecular</subject><subject>Molecular replacement</subject><subject>Mutation</subject><subject>Protein Conformation</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Sf9 Cells</subject><subject>Technical Note</subject><subject>X-ray crystallography</subject><issn>1047-8477</issn><issn>1095-8657</issn><issn>1095-8657</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>D8T</sourceid><recordid>eNp9kk1v1DAQhiMEakvpD-CCcuSSxRM7diIkJFSVD6kSFzgiy3Emu14SO9hJy_57ZrXb0h6KZNnj8fO-Hmkmy14DWwED-W672qZ2VVK4YkBLPsvOgDVVUctKPd_HQhW1UOo0e5nSljEmoIST7LSUdcVAsbPs55VJu3w042gGZ3yOf6aIKbngc-O73MZdms0whHU002aXh57YYQ4Ji9b5zvl1PsUwo_NFvyTs8s0ykssxl15lL3ozJLw4nufZj09X3y-_FNffPn-9_HhdWMmqmfbOsFIoVKZuFZVWlshrzitluADBVds0DSjEHoTg1jbSCLobWYEUrGv4eVYcfNMtTkurp-hGE3c6GKePqV8Uoa6YEhUQ3zzJU-3dP9GdEDhnjay5IO2Hg5aAETuLfo5meGzx6MW7jV6HG019gFoxMnh7NIjh94Jp1qNLFofBeAxL0qCkkmXZSEkoHFAbQ0oR-_tvgOn9COitphHQ-xHQDGjtNW8e1nevuOs5Ae8PAFJHbhxGnaxDb7FzEe2su-D-Y_8XambE2g</recordid><startdate>20160401</startdate><enddate>20160401</enddate><creator>Bokhove, Marcel</creator><creator>Sadat Al Hosseini, Hamed</creator><creator>Saito, Takako</creator><creator>Dioguardi, Elisa</creator><creator>Gegenschatz-Schmid, Katharina</creator><creator>Nishimura, Kaoru</creator><creator>Raj, Isha</creator><creator>de Sanctis, Daniele</creator><creator>Han, Ling</creator><creator>Jovine, Luca</creator><general>Elsevier Inc</general><general>Academic Press</general><scope>6I.</scope><scope>AAFTH</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>5PM</scope><scope>ADTPV</scope><scope>AOWAS</scope><scope>D8T</scope><scope>ZZAVC</scope><orcidid>https://orcid.org/0000-0002-2679-6946</orcidid><orcidid>https://orcid.org/0000-0001-7241-5967</orcidid></search><sort><creationdate>20160401</creationdate><title>Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins</title><author>Bokhove, Marcel ; Sadat Al Hosseini, Hamed ; Saito, Takako ; Dioguardi, Elisa ; Gegenschatz-Schmid, Katharina ; Nishimura, Kaoru ; Raj, Isha ; de Sanctis, Daniele ; Han, Ling ; Jovine, Luca</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c605t-c6da0247e7a8b785022e383357a341437b99917eef1443cc96a4917a651640d93</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Base Sequence</topic><topic>CHO Cells</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Crystallization</topic><topic>Crystallography, X-Ray</topic><topic>Gene Expression</topic><topic>Glycoproteins</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Maltose-binding protein fusion</topic><topic>Maltose-Binding Proteins - chemistry</topic><topic>Maltose-Binding Proteins - genetics</topic><topic>Maltose-Binding Proteins - metabolism</topic><topic>Mammalian cell expression</topic><topic>Medicin och hälsovetenskap</topic><topic>Models, Molecular</topic><topic>Molecular replacement</topic><topic>Mutation</topic><topic>Protein Conformation</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Sf9 Cells</topic><topic>Technical Note</topic><topic>X-ray crystallography</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bokhove, Marcel</creatorcontrib><creatorcontrib>Sadat Al Hosseini, Hamed</creatorcontrib><creatorcontrib>Saito, Takako</creatorcontrib><creatorcontrib>Dioguardi, Elisa</creatorcontrib><creatorcontrib>Gegenschatz-Schmid, Katharina</creatorcontrib><creatorcontrib>Nishimura, Kaoru</creatorcontrib><creatorcontrib>Raj, Isha</creatorcontrib><creatorcontrib>de Sanctis, Daniele</creatorcontrib><creatorcontrib>Han, Ling</creatorcontrib><creatorcontrib>Jovine, Luca</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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>PubMed Central (Full Participant titles)</collection><collection>SwePub</collection><collection>SwePub Articles</collection><collection>SWEPUB Freely available online</collection><collection>SwePub Articles full text</collection><jtitle>Journal of structural biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bokhove, Marcel</au><au>Sadat Al Hosseini, Hamed</au><au>Saito, Takako</au><au>Dioguardi, Elisa</au><au>Gegenschatz-Schmid, Katharina</au><au>Nishimura, Kaoru</au><au>Raj, Isha</au><au>de Sanctis, Daniele</au><au>Han, Ling</au><au>Jovine, Luca</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins</atitle><jtitle>Journal of structural biology</jtitle><addtitle>J Struct Biol</addtitle><date>2016-04-01</date><risdate>2016</risdate><volume>194</volume><issue>1</issue><spage>1</spage><epage>7</epage><pages>1-7</pages><issn>1047-8477</issn><issn>1095-8657</issn><eissn>1095-8657</eissn><abstract>We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>26850170</pmid><doi>10.1016/j.jsb.2016.01.016</doi><tpages>7</tpages><orcidid>https://orcid.org/0000-0002-2679-6946</orcidid><orcidid>https://orcid.org/0000-0001-7241-5967</orcidid><oa>free_for_read</oa></addata></record> |
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| language | eng |
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| subjects | Amino Acid Sequence Animals Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Base Sequence CHO Cells Cricetinae Cricetulus Crystallization Crystallography, X-Ray Gene Expression Glycoproteins HEK293 Cells Humans Maltose-binding protein fusion Maltose-Binding Proteins - chemistry Maltose-Binding Proteins - genetics Maltose-Binding Proteins - metabolism Mammalian cell expression Medicin och hälsovetenskap Models, Molecular Molecular replacement Mutation Protein Conformation Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Sf9 Cells Technical Note X-ray crystallography |
| title | Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins |
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