Purification of the large ribosomal subunit via its association with the small subunit
We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) t...
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| Veröffentlicht in: | Analytical biochemistry 2009-12, Vol.395 (1), p.77-85 |
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| creator | Simons, Samuel P. McLellan, Thomas J. Aeed, Paul A. Zaniewski, Richard P. Desbonnet, Charlene R. Wondrack, Lillian M. Marr, Eric S. Subashi, Timothy A. Dougherty, Thomas J. Xu, Zuoyu Wang, Ing-Kae LeMotte, Peter K. Maguire, Bruce A. |
| description | We have developed an affinity purification of the large ribosomal subunit from
Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity. |
| doi_str_mv | 10.1016/j.ab.2009.07.042 |
| format | Article |
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Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/j.ab.2009.07.042</identifier><identifier>PMID: 19646947</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>50S subunit ; Affinity purification ; Bacterial Proteins - analysis ; Centrifugation, Density Gradient ; Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Cloning, Molecular ; Complex purification ; Databases, Protein ; Deinococcus - ultrastructure ; Deinococcus radiodurans ; Gene Expression ; Loose couples ; Magnesium Chloride ; Oligopeptides ; Peptide Fragments - analysis ; Peptides - genetics ; Recombinant Fusion Proteins ; Ribosomal Proteins - analysis ; Ribosomal Proteins - genetics ; Ribosome ; Ribosome Subunits, Large, Bacterial - chemistry ; Ribosome Subunits, Large, Bacterial - metabolism ; Ribosome Subunits, Small, Bacterial - genetics ; Ribosome Subunits, Small, Bacterial - metabolism ; RNA, Bacterial - analysis ; RNA, Ribosomal - analysis ; Spectrometry, Mass, Electrospray Ionization ; Subunit association ; Tandem Mass Spectrometry ; Tight couples</subject><ispartof>Analytical biochemistry, 2009-12, Vol.395 (1), p.77-85</ispartof><rights>2009 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c380t-f673a1b0aae279a1ff1e31b038ab309bf5e69b19bf45ea6b423e48a0db949ea03</citedby><cites>FETCH-LOGICAL-c380t-f673a1b0aae279a1ff1e31b038ab309bf5e69b19bf45ea6b423e48a0db949ea03</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0003269709005284$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19646947$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Simons, Samuel P.</creatorcontrib><creatorcontrib>McLellan, Thomas J.</creatorcontrib><creatorcontrib>Aeed, Paul A.</creatorcontrib><creatorcontrib>Zaniewski, Richard P.</creatorcontrib><creatorcontrib>Desbonnet, Charlene R.</creatorcontrib><creatorcontrib>Wondrack, Lillian M.</creatorcontrib><creatorcontrib>Marr, Eric S.</creatorcontrib><creatorcontrib>Subashi, Timothy A.</creatorcontrib><creatorcontrib>Dougherty, Thomas J.</creatorcontrib><creatorcontrib>Xu, Zuoyu</creatorcontrib><creatorcontrib>Wang, Ing-Kae</creatorcontrib><creatorcontrib>LeMotte, Peter K.</creatorcontrib><creatorcontrib>Maguire, Bruce A.</creatorcontrib><title>Purification of the large ribosomal subunit via its association with the small subunit</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>We have developed an affinity purification of the large ribosomal subunit from
Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.</description><subject>50S subunit</subject><subject>Affinity purification</subject><subject>Bacterial Proteins - analysis</subject><subject>Centrifugation, Density Gradient</subject><subject>Chromatography, Affinity</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Cloning, Molecular</subject><subject>Complex purification</subject><subject>Databases, Protein</subject><subject>Deinococcus - ultrastructure</subject><subject>Deinococcus radiodurans</subject><subject>Gene Expression</subject><subject>Loose couples</subject><subject>Magnesium Chloride</subject><subject>Oligopeptides</subject><subject>Peptide Fragments - analysis</subject><subject>Peptides - genetics</subject><subject>Recombinant Fusion Proteins</subject><subject>Ribosomal Proteins - analysis</subject><subject>Ribosomal Proteins - genetics</subject><subject>Ribosome</subject><subject>Ribosome Subunits, Large, Bacterial - chemistry</subject><subject>Ribosome Subunits, Large, Bacterial - metabolism</subject><subject>Ribosome Subunits, Small, Bacterial - genetics</subject><subject>Ribosome Subunits, Small, Bacterial - metabolism</subject><subject>RNA, Bacterial - analysis</subject><subject>RNA, Ribosomal - analysis</subject><subject>Spectrometry, Mass, Electrospray Ionization</subject><subject>Subunit association</subject><subject>Tandem Mass Spectrometry</subject><subject>Tight couples</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kE1LxDAQhoMoun7cPUlPemqdNGnaeBPxCwQ9qNcw6U41S3ejSav4743uoic9zQw87wvzMLbPoeDA1fGsQFuUALqAugBZrrEJB61yEKDX2QQARF4qXW-x7RhnAJzLSm2yLa6VVFrWE_Z4NwbXuRYH5xeZ77LhmbIewxNlwVkf_Rz7LI52XLghe3OYuSFmGKNv3TLy7obn71BM5A-6yzY67CPtreYOe7g4vz-7ym9uL6_PTm_yVjQw5J2qBXILiFTWGnnXcRLpFg3a9ILtKlLa8rTIilBZWQqSDcLUaqkJQeywo2XvS_CvI8XBzF1sqe9xQX6MphYSqqppdCIP_yVLDo3WgicQlmAbfIyBOvMS3BzDh-FgvqybmUFrvqwbqE2yniIHq-7Rzmn6G1hpTsDJEqDk4s1RMLF1tGhp6gK1g5l693f7J_WRkpg</recordid><startdate>20091201</startdate><enddate>20091201</enddate><creator>Simons, Samuel P.</creator><creator>McLellan, Thomas J.</creator><creator>Aeed, Paul A.</creator><creator>Zaniewski, Richard P.</creator><creator>Desbonnet, Charlene R.</creator><creator>Wondrack, Lillian M.</creator><creator>Marr, Eric S.</creator><creator>Subashi, Timothy A.</creator><creator>Dougherty, Thomas J.</creator><creator>Xu, Zuoyu</creator><creator>Wang, Ing-Kae</creator><creator>LeMotte, Peter K.</creator><creator>Maguire, Bruce A.</creator><general>Elsevier Inc</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>7QL</scope><scope>7TM</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>20091201</creationdate><title>Purification of the large ribosomal subunit via its association with the small subunit</title><author>Simons, Samuel P. ; McLellan, Thomas J. ; Aeed, Paul A. ; Zaniewski, Richard P. ; Desbonnet, Charlene R. ; Wondrack, Lillian M. ; Marr, Eric S. ; Subashi, Timothy A. ; Dougherty, Thomas J. ; Xu, Zuoyu ; Wang, Ing-Kae ; LeMotte, Peter K. ; Maguire, Bruce A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c380t-f673a1b0aae279a1ff1e31b038ab309bf5e69b19bf45ea6b423e48a0db949ea03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>50S subunit</topic><topic>Affinity purification</topic><topic>Bacterial Proteins - analysis</topic><topic>Centrifugation, Density Gradient</topic><topic>Chromatography, Affinity</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Cloning, Molecular</topic><topic>Complex purification</topic><topic>Databases, Protein</topic><topic>Deinococcus - ultrastructure</topic><topic>Deinococcus radiodurans</topic><topic>Gene Expression</topic><topic>Loose couples</topic><topic>Magnesium Chloride</topic><topic>Oligopeptides</topic><topic>Peptide Fragments - analysis</topic><topic>Peptides - genetics</topic><topic>Recombinant Fusion Proteins</topic><topic>Ribosomal Proteins - analysis</topic><topic>Ribosomal Proteins - genetics</topic><topic>Ribosome</topic><topic>Ribosome Subunits, Large, Bacterial - chemistry</topic><topic>Ribosome Subunits, Large, Bacterial - metabolism</topic><topic>Ribosome Subunits, Small, Bacterial - genetics</topic><topic>Ribosome Subunits, Small, Bacterial - metabolism</topic><topic>RNA, Bacterial - analysis</topic><topic>RNA, Ribosomal - analysis</topic><topic>Spectrometry, Mass, Electrospray Ionization</topic><topic>Subunit association</topic><topic>Tandem Mass Spectrometry</topic><topic>Tight couples</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Simons, Samuel P.</creatorcontrib><creatorcontrib>McLellan, Thomas J.</creatorcontrib><creatorcontrib>Aeed, Paul A.</creatorcontrib><creatorcontrib>Zaniewski, Richard P.</creatorcontrib><creatorcontrib>Desbonnet, Charlene R.</creatorcontrib><creatorcontrib>Wondrack, Lillian M.</creatorcontrib><creatorcontrib>Marr, Eric S.</creatorcontrib><creatorcontrib>Subashi, Timothy A.</creatorcontrib><creatorcontrib>Dougherty, Thomas J.</creatorcontrib><creatorcontrib>Xu, Zuoyu</creatorcontrib><creatorcontrib>Wang, Ing-Kae</creatorcontrib><creatorcontrib>LeMotte, Peter K.</creatorcontrib><creatorcontrib>Maguire, Bruce A.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Simons, Samuel P.</au><au>McLellan, Thomas J.</au><au>Aeed, Paul A.</au><au>Zaniewski, Richard P.</au><au>Desbonnet, Charlene R.</au><au>Wondrack, Lillian M.</au><au>Marr, Eric S.</au><au>Subashi, Timothy A.</au><au>Dougherty, Thomas J.</au><au>Xu, Zuoyu</au><au>Wang, Ing-Kae</au><au>LeMotte, Peter K.</au><au>Maguire, Bruce A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification of the large ribosomal subunit via its association with the small subunit</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2009-12-01</date><risdate>2009</risdate><volume>395</volume><issue>1</issue><spage>77</spage><epage>85</epage><pages>77-85</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>We have developed an affinity purification of the large ribosomal subunit from
Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>19646947</pmid><doi>10.1016/j.ab.2009.07.042</doi><tpages>9</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 2009-12, Vol.395 (1), p.77-85 |
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| subjects | 50S subunit Affinity purification Bacterial Proteins - analysis Centrifugation, Density Gradient Chromatography, Affinity Chromatography, High Pressure Liquid Cloning, Molecular Complex purification Databases, Protein Deinococcus - ultrastructure Deinococcus radiodurans Gene Expression Loose couples Magnesium Chloride Oligopeptides Peptide Fragments - analysis Peptides - genetics Recombinant Fusion Proteins Ribosomal Proteins - analysis Ribosomal Proteins - genetics Ribosome Ribosome Subunits, Large, Bacterial - chemistry Ribosome Subunits, Large, Bacterial - metabolism Ribosome Subunits, Small, Bacterial - genetics Ribosome Subunits, Small, Bacterial - metabolism RNA, Bacterial - analysis RNA, Ribosomal - analysis Spectrometry, Mass, Electrospray Ionization Subunit association Tandem Mass Spectrometry Tight couples |
| title | Purification of the large ribosomal subunit via its association with the small subunit |
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