Reconstitution of Purified Bacterial Preprotein Translocase in Liposomes
Protein translocation is one of the examples of a complex membrane-bound biological process that has been functionally reconstituted into proteoliposomes. This process relies on several membrane proteins that need to be coreconstituted to yield a functional system. This chapter describes methods to...
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| Veröffentlicht in: | Methods in Enzymology 2003, Vol.372, p.86-98 |
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| creator | Does, Chris van der Keyzer, Jeanine de Laan, Martin van der Driessen, Arnold J.M |
| description | Protein translocation is one of the examples of a complex membrane-bound biological process that has been functionally reconstituted into proteoliposomes. This process relies on several membrane proteins that need to be coreconstituted to yield a functional system. This chapter describes methods to express, detergent solubilize, purify, and functionally reconstitute preprotein translocation in proteoliposomes using Escherichia coli translocase components. A coreconstitution protocol and a fluorescence assay to monitor preprotein translocation in vitro are described. The proteins can be solubilized from the membrane and purified to homogeneity (>95%) by anion exchange and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. A multitude of methods are described for the reconstitution of membrane proteins into proteoliposomes. The methods of hydrophobic absorption or rapid dilution for detergent removal are the most significant methods for the functional reconstitution. The functional-membrane integration of newly synthesized inner membrane proteins, using the purified translocase and associated components, remains a challenge for future research. Other challenges lie in the reconstitution of the complete assembly of multisubunit membrane proteins. |
| doi_str_mv | 10.1016/S0076-6879(03)72005-9 |
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The functional-membrane integration of newly synthesized inner membrane proteins, using the purified translocase and associated components, remains a challenge for future research. Other challenges lie in the reconstitution of the complete assembly of multisubunit membrane proteins.</description><identifier>ISSN: 0076-6879</identifier><identifier>ISBN: 9780121822750</identifier><identifier>ISBN: 0121822753</identifier><identifier>EISSN: 1557-7988</identifier><identifier>DOI: 10.1016/S0076-6879(03)72005-9</identifier><identifier>PMID: 14610808</identifier><language>eng</language><publisher>United States: Elsevier Science & Technology</publisher><subject>Adenosine Triphosphatases - genetics ; Adenosine Triphosphatases - isolation & purification ; Adenosine Triphosphatases - metabolism ; Bacterial Outer Membrane Proteins - metabolism ; Bacterial Proteins ; Dimerization ; Electrophoresis, Polyacrylamide Gel - methods ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - isolation & purification ; Escherichia coli Proteins - metabolism ; Kinetics ; Membrane Proteins - isolation & purification ; Membrane Proteins - metabolism ; Membrane Transport Proteins - genetics ; Membrane Transport Proteins - isolation & purification ; Membrane Transport Proteins - metabolism ; Protein Precursors - metabolism ; SEC Translocation Channels</subject><ispartof>Methods in Enzymology, 2003, Vol.372, p.86-98</ispartof><rights>2003 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c367t-8d070f6243905fe7cf67bb686c87c176b5a78975ccd46a4329eb4afeaff217263</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0076687903720059$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,775,776,780,789,3446,3537,4010,11267,27900,27901,27902,45786,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14610808$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Does, Chris van der</creatorcontrib><creatorcontrib>Keyzer, Jeanine de</creatorcontrib><creatorcontrib>Laan, Martin van der</creatorcontrib><creatorcontrib>Driessen, Arnold J.M</creatorcontrib><title>Reconstitution of Purified Bacterial Preprotein Translocase in Liposomes</title><title>Methods in Enzymology</title><addtitle>Methods Enzymol</addtitle><description>Protein translocation is one of the examples of a complex membrane-bound biological process that has been functionally reconstituted into proteoliposomes. This process relies on several membrane proteins that need to be coreconstituted to yield a functional system. This chapter describes methods to express, detergent solubilize, purify, and functionally reconstitute preprotein translocation in proteoliposomes using Escherichia coli translocase components. A coreconstitution protocol and a fluorescence assay to monitor preprotein translocation in vitro are described. The proteins can be solubilized from the membrane and purified to homogeneity (>95%) by anion exchange and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. A multitude of methods are described for the reconstitution of membrane proteins into proteoliposomes. The methods of hydrophobic absorption or rapid dilution for detergent removal are the most significant methods for the functional reconstitution. The functional-membrane integration of newly synthesized inner membrane proteins, using the purified translocase and associated components, remains a challenge for future research. Other challenges lie in the reconstitution of the complete assembly of multisubunit membrane proteins.</description><subject>Adenosine Triphosphatases - genetics</subject><subject>Adenosine Triphosphatases - isolation & purification</subject><subject>Adenosine Triphosphatases - metabolism</subject><subject>Bacterial Outer Membrane Proteins - metabolism</subject><subject>Bacterial Proteins</subject><subject>Dimerization</subject><subject>Electrophoresis, Polyacrylamide Gel - methods</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - isolation & purification</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Kinetics</subject><subject>Membrane Proteins - isolation & purification</subject><subject>Membrane Proteins - metabolism</subject><subject>Membrane Transport Proteins - genetics</subject><subject>Membrane Transport Proteins - isolation & purification</subject><subject>Membrane Transport Proteins - metabolism</subject><subject>Protein Precursors - metabolism</subject><subject>SEC Translocation Channels</subject><issn>0076-6879</issn><issn>1557-7988</issn><isbn>9780121822750</isbn><isbn>0121822753</isbn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kF1L5TAQhoMf6Fn1Jyi9Er3o7iRpM-mVqOgqHFjZda9Dmk4g0tMck1bw39vj19Uw8LzDOw9jxxx-cuDq1z8AVKXS2JyBPEcBUJfNFlvwusYSG6232VGDGrjgWgisYYctviP77EfOTwACdcP32D6vFAcNesHu_pKLQx7DOI0hDkX0xcOUgg_UFVfWjZSC7YuHROsURwpD8ZjskPvobKZiXpdhHXNcUT5ku972mY4-5wH7f3vzeH1XLv_8vr--XJZOKhxL3QGCV6KSDdSe0HmFbau0chodR9XWdu6ItXNdpWwlRUNtZT1Z7wVHoeQBO_24Oxd6niiPZhWyo763A8UpG-Sy0lJvwJNPcGpX1Jl1CiubXs3X7zNw8QHQXPclUDLZBRocdSGRG00Xg-FgNvbNu32zUWlAmnf7ppFvzQpz3Q</recordid><startdate>2003</startdate><enddate>2003</enddate><creator>Does, Chris van der</creator><creator>Keyzer, Jeanine de</creator><creator>Laan, Martin van der</creator><creator>Driessen, Arnold J.M</creator><general>Elsevier Science & Technology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>2003</creationdate><title>Reconstitution of Purified Bacterial Preprotein Translocase in Liposomes</title><author>Does, Chris van der ; Keyzer, Jeanine de ; Laan, Martin van der ; Driessen, Arnold J.M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c367t-8d070f6243905fe7cf67bb686c87c176b5a78975ccd46a4329eb4afeaff217263</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Adenosine Triphosphatases - genetics</topic><topic>Adenosine Triphosphatases - isolation & purification</topic><topic>Adenosine Triphosphatases - metabolism</topic><topic>Bacterial Outer Membrane Proteins - metabolism</topic><topic>Bacterial Proteins</topic><topic>Dimerization</topic><topic>Electrophoresis, Polyacrylamide Gel - methods</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - isolation & purification</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Kinetics</topic><topic>Membrane Proteins - isolation & purification</topic><topic>Membrane Proteins - metabolism</topic><topic>Membrane Transport Proteins - genetics</topic><topic>Membrane Transport Proteins - isolation & purification</topic><topic>Membrane Transport Proteins - metabolism</topic><topic>Protein Precursors - metabolism</topic><topic>SEC Translocation Channels</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Does, Chris van der</creatorcontrib><creatorcontrib>Keyzer, Jeanine de</creatorcontrib><creatorcontrib>Laan, Martin van der</creatorcontrib><creatorcontrib>Driessen, Arnold J.M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>Methods in Enzymology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Does, Chris van der</au><au>Keyzer, Jeanine de</au><au>Laan, Martin van der</au><au>Driessen, Arnold J.M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Reconstitution of Purified Bacterial Preprotein Translocase in Liposomes</atitle><jtitle>Methods in Enzymology</jtitle><addtitle>Methods Enzymol</addtitle><date>2003</date><risdate>2003</risdate><volume>372</volume><spage>86</spage><epage>98</epage><pages>86-98</pages><issn>0076-6879</issn><eissn>1557-7988</eissn><isbn>9780121822750</isbn><isbn>0121822753</isbn><abstract>Protein translocation is one of the examples of a complex membrane-bound biological process that has been functionally reconstituted into proteoliposomes. This process relies on several membrane proteins that need to be coreconstituted to yield a functional system. This chapter describes methods to express, detergent solubilize, purify, and functionally reconstitute preprotein translocation in proteoliposomes using Escherichia coli translocase components. A coreconstitution protocol and a fluorescence assay to monitor preprotein translocation in vitro are described. The proteins can be solubilized from the membrane and purified to homogeneity (>95%) by anion exchange and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. A multitude of methods are described for the reconstitution of membrane proteins into proteoliposomes. The methods of hydrophobic absorption or rapid dilution for detergent removal are the most significant methods for the functional reconstitution. 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| subjects | Adenosine Triphosphatases - genetics Adenosine Triphosphatases - isolation & purification Adenosine Triphosphatases - metabolism Bacterial Outer Membrane Proteins - metabolism Bacterial Proteins Dimerization Electrophoresis, Polyacrylamide Gel - methods Escherichia coli Proteins - genetics Escherichia coli Proteins - isolation & purification Escherichia coli Proteins - metabolism Kinetics Membrane Proteins - isolation & purification Membrane Proteins - metabolism Membrane Transport Proteins - genetics Membrane Transport Proteins - isolation & purification Membrane Transport Proteins - metabolism Protein Precursors - metabolism SEC Translocation Channels |
| title | Reconstitution of Purified Bacterial Preprotein Translocase in Liposomes |
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