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Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin
The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 °C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fus...
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| Published in: | Analytical biochemistry 2006-09, Vol.356 (1), p.86-93 |
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| Main Authors: | , , , |
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| cites | cdi_FETCH-LOGICAL-c379t-53432355eea6173bf9d854506561efdba304cd4744dbd99c1b5e27b7fa9768a63 |
| container_end_page | 93 |
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| container_title | Analytical biochemistry |
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| creator | Mills, Kenneth V. Connor, Katherine R. Dorval, Deirdre M. Lewandowski, Katherine T. |
| description | The intein that interrupts the DNA polymerase II DP2 subunit in
Pyrococcus abyssi can be overexpressed in
Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37
°C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40
°C or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage. |
| doi_str_mv | 10.1016/j.ab.2006.04.055 |
| format | article |
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Pyrococcus abyssi can be overexpressed in
Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37
°C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40
°C or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/j.ab.2006.04.055</identifier><identifier>PMID: 16756933</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Affinity Labels ; Base Sequence ; DNA Polymerase II - genetics ; DNA Polymerase II - isolation & purification ; DNA, Archaeal - genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Escherichia coli - genetics ; In Vitro Techniques ; Intein ; Inteins ; Plasmids - genetics ; Protein purification ; Protein Splicing ; Proteins - genetics ; Proteins - isolation & purification ; Pyrococcus abyssi ; Pyrococcus abyssi - enzymology ; Pyrococcus abyssi - genetics ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Resins, Synthetic ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Temperature</subject><ispartof>Analytical biochemistry, 2006-09, Vol.356 (1), p.86-93</ispartof><rights>2006 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c379t-53432355eea6173bf9d854506561efdba304cd4744dbd99c1b5e27b7fa9768a63</citedby><cites>FETCH-LOGICAL-c379t-53432355eea6173bf9d854506561efdba304cd4744dbd99c1b5e27b7fa9768a63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,27957,27958</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16756933$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Mills, Kenneth V.</creatorcontrib><creatorcontrib>Connor, Katherine R.</creatorcontrib><creatorcontrib>Dorval, Deirdre M.</creatorcontrib><creatorcontrib>Lewandowski, Katherine T.</creatorcontrib><title>Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>The intein that interrupts the DNA polymerase II DP2 subunit in
Pyrococcus abyssi can be overexpressed in
Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37
°C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40
°C or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.</description><subject>Affinity Labels</subject><subject>Base Sequence</subject><subject>DNA Polymerase II - genetics</subject><subject>DNA Polymerase II - isolation & purification</subject><subject>DNA, Archaeal - genetics</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>In Vitro Techniques</subject><subject>Intein</subject><subject>Inteins</subject><subject>Plasmids - genetics</subject><subject>Protein purification</subject><subject>Protein Splicing</subject><subject>Proteins - genetics</subject><subject>Proteins - isolation & purification</subject><subject>Pyrococcus abyssi</subject><subject>Pyrococcus abyssi - enzymology</subject><subject>Pyrococcus abyssi - genetics</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Resins, Synthetic</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><subject>Temperature</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><recordid>eNqFkT2P1DAURS0EYmcXeirkioqE53Fsx3RoBQvSSlBAbfnjBXmUOFnbGWn315PRjESFqF7xzr3FPYS8YdAyYPLDobWu3QPIFroWhHhGdgy0bICDfk52AMCbvdTqilyXcgBgrBPyJbliUgmpOd-Rhx95rhgTXdYch-htjXOix2hpxWnBbOuasQm4YAqY6nsa0wlvJgzRVgzUj2iP9jfSIc8TtYnGaZpdHOPT9pyw2pHaYYgp1keascT0irwY7Fjw9eXekF9fPv-8_drcf7_7dvvpvvFc6doI3vE9FwLRSqa4G3ToRSdACslwCM5y6HzoVNcFF7T2zAncK6cGq5XsreQ35N25d8nzw4qlmikWj-NoE85rMbJXUmj4P8i00j3vTyCcQZ_nUjIOZslxsvnRMDAnH-ZgrDMnHwY6s_nYIm8v3avbJvsbuAjYgI9nALcpjhGzKT5i8tu8GX01YY7_bv8D2X-cKg</recordid><startdate>20060901</startdate><enddate>20060901</enddate><creator>Mills, Kenneth V.</creator><creator>Connor, Katherine R.</creator><creator>Dorval, Deirdre M.</creator><creator>Lewandowski, Katherine T.</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>7QO</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20060901</creationdate><title>Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin</title><author>Mills, Kenneth V. ; Connor, Katherine R. ; Dorval, Deirdre M. ; Lewandowski, Katherine T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c379t-53432355eea6173bf9d854506561efdba304cd4744dbd99c1b5e27b7fa9768a63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Affinity Labels</topic><topic>Base Sequence</topic><topic>DNA Polymerase II - genetics</topic><topic>DNA Polymerase II - isolation & purification</topic><topic>DNA, Archaeal - genetics</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>In Vitro Techniques</topic><topic>Intein</topic><topic>Inteins</topic><topic>Plasmids - genetics</topic><topic>Protein purification</topic><topic>Protein Splicing</topic><topic>Proteins - genetics</topic><topic>Proteins - isolation & purification</topic><topic>Pyrococcus abyssi</topic><topic>Pyrococcus abyssi - enzymology</topic><topic>Pyrococcus abyssi - genetics</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Resins, Synthetic</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mills, Kenneth V.</creatorcontrib><creatorcontrib>Connor, Katherine R.</creatorcontrib><creatorcontrib>Dorval, Deirdre M.</creatorcontrib><creatorcontrib>Lewandowski, Katherine T.</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>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mills, Kenneth V.</au><au>Connor, Katherine R.</au><au>Dorval, Deirdre M.</au><au>Lewandowski, Katherine T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2006-09-01</date><risdate>2006</risdate><volume>356</volume><issue>1</issue><spage>86</spage><epage>93</epage><pages>86-93</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>The intein that interrupts the DNA polymerase II DP2 subunit in
Pyrococcus abyssi can be overexpressed in
Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37
°C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40
°C or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>16756933</pmid><doi>10.1016/j.ab.2006.04.055</doi><tpages>8</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 2006-09, Vol.356 (1), p.86-93 |
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| source | ScienceDirect Freedom Collection |
| subjects | Affinity Labels Base Sequence DNA Polymerase II - genetics DNA Polymerase II - isolation & purification DNA, Archaeal - genetics Electrophoresis, Polyacrylamide Gel Escherichia coli Escherichia coli - genetics In Vitro Techniques Intein Inteins Plasmids - genetics Protein purification Protein Splicing Proteins - genetics Proteins - isolation & purification Pyrococcus abyssi Pyrococcus abyssi - enzymology Pyrococcus abyssi - genetics Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Resins, Synthetic Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Temperature |
| title | Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin |
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