A Procedure for Renaturation and Purification of the Extracellular Serratia marcescens Nuclease from Genetically Engineered Escherichia coli
Overproduction of the extracellular Serratia marcescens nuclease in Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedure...
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| Veröffentlicht in: | Protein expression and purification 1994-02, Vol.5 (1), p.37-43 |
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| container_title | Protein expression and purification |
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| creator | Friedhoff, P. Gimadutdinow, O. Ruter, T. Wende, W. Urbanke, C. Thole, H. Pingoud, A. |
| description | Overproduction of the extracellular
Serratia marcescens nuclease in
Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedures. The cell-associated insoluble protein can be solubilized in 6 M urea after breaking up the cells by sonication. Renaturation is achieved by dilution or dialysis. Subsequent phosphocellulose chromatography yields a homogeneous protein preparation which is shown by a variety of biochemical and biophysical analyses to be indistinguishable from conventionally prepared material. The high yield (>10 mg/500-ml culture) and the ease of preparation (2 to 3 days) make this an attractive alternative to previously described procedures. |
| doi_str_mv | 10.1006/prep.1994.1005 |
| format | Article |
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Serratia marcescens nuclease in
Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedures. The cell-associated insoluble protein can be solubilized in 6 M urea after breaking up the cells by sonication. Renaturation is achieved by dilution or dialysis. Subsequent phosphocellulose chromatography yields a homogeneous protein preparation which is shown by a variety of biochemical and biophysical analyses to be indistinguishable from conventionally prepared material. The high yield (>10 mg/500-ml culture) and the ease of preparation (2 to 3 days) make this an attractive alternative to previously described procedures.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1006/prep.1994.1005</identifier><identifier>PMID: 8167472</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amino Acid Sequence ; Bacterial Proteins - biosynthesis ; Bacterial Proteins - isolation & purification ; Cellulose - analogs & derivatives ; Chromatography, Affinity ; Endodeoxyribonucleases - biosynthesis ; Endodeoxyribonucleases - isolation & purification ; Endoribonucleases - biosynthesis ; Endoribonucleases - isolation & purification ; Escherichia coli ; Protein Denaturation ; Protein Folding ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - isolation & purification ; Serratia marcescens - enzymology ; Serratia marcescens - genetics ; Urea</subject><ispartof>Protein expression and purification, 1994-02, Vol.5 (1), p.37-43</ispartof><rights>1994 Academic Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c405t-796aad0db845bce6ed6e547261a12ed558550aa76c15ae675c65ca44882b54b43</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/prep.1994.1005$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3549,27923,27924,45994</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8167472$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Friedhoff, P.</creatorcontrib><creatorcontrib>Gimadutdinow, O.</creatorcontrib><creatorcontrib>Ruter, T.</creatorcontrib><creatorcontrib>Wende, W.</creatorcontrib><creatorcontrib>Urbanke, C.</creatorcontrib><creatorcontrib>Thole, H.</creatorcontrib><creatorcontrib>Pingoud, A.</creatorcontrib><title>A Procedure for Renaturation and Purification of the Extracellular Serratia marcescens Nuclease from Genetically Engineered Escherichia coli</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>Overproduction of the extracellular
Serratia marcescens nuclease in
Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedures. The cell-associated insoluble protein can be solubilized in 6 M urea after breaking up the cells by sonication. Renaturation is achieved by dilution or dialysis. Subsequent phosphocellulose chromatography yields a homogeneous protein preparation which is shown by a variety of biochemical and biophysical analyses to be indistinguishable from conventionally prepared material. The high yield (>10 mg/500-ml culture) and the ease of preparation (2 to 3 days) make this an attractive alternative to previously described procedures.</description><subject>Amino Acid Sequence</subject><subject>Bacterial Proteins - biosynthesis</subject><subject>Bacterial Proteins - isolation & purification</subject><subject>Cellulose - analogs & derivatives</subject><subject>Chromatography, Affinity</subject><subject>Endodeoxyribonucleases - biosynthesis</subject><subject>Endodeoxyribonucleases - isolation & purification</subject><subject>Endoribonucleases - biosynthesis</subject><subject>Endoribonucleases - isolation & purification</subject><subject>Escherichia coli</subject><subject>Protein Denaturation</subject><subject>Protein Folding</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Serratia marcescens - enzymology</subject><subject>Serratia marcescens - genetics</subject><subject>Urea</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kEtv1DAUhS0EKqWwZYfkFbsMdupHsqyqoa1UQcVjbd3YN4xRxh6uE0T_Az-6jmbEjtV9nftJ5zD2VoqNFMJ8OBAeNrLv1TrqZ-xcit40orX987VXptF9271kr0r5KYSURugzdtZJY5Vtz9nfK_5A2WNYCPmYiX_BBPNCMMecOKTAHxaKY_THRR75vEO-_TMTeJymZQLiX5FWPfA9kMfiMRX-afETQqlMynt-gwnnypimR75NP2JCJAx8W_wOKfpd_fV5iq_ZixGmgm9O9YJ9_7j9dn3b3H--ubu-um-8EnpubG8AgghDp_Tg0WAwqKsbI0G2GLTutBYA1nipAY3V3mgPSnVdO2g1qMsL9v7IPVD-tWCZ3T6W1Q4kzEtx1ijVW9tW4eYo9JRLIRzdgWJ1-eikcGv8bo3frfGvo64P707kZdhj-Cc_5V3v3fGO1d7viOSKj5hq_pHQzy7k-D_0E132lyI</recordid><startdate>19940201</startdate><enddate>19940201</enddate><creator>Friedhoff, P.</creator><creator>Gimadutdinow, O.</creator><creator>Ruter, T.</creator><creator>Wende, W.</creator><creator>Urbanke, C.</creator><creator>Thole, H.</creator><creator>Pingoud, 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>7X8</scope></search><sort><creationdate>19940201</creationdate><title>A Procedure for Renaturation and Purification of the Extracellular Serratia marcescens Nuclease from Genetically Engineered Escherichia coli</title><author>Friedhoff, P. ; 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Serratia marcescens nuclease in
Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedures. The cell-associated insoluble protein can be solubilized in 6 M urea after breaking up the cells by sonication. Renaturation is achieved by dilution or dialysis. Subsequent phosphocellulose chromatography yields a homogeneous protein preparation which is shown by a variety of biochemical and biophysical analyses to be indistinguishable from conventionally prepared material. The high yield (>10 mg/500-ml culture) and the ease of preparation (2 to 3 days) make this an attractive alternative to previously described procedures.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>8167472</pmid><doi>10.1006/prep.1994.1005</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1046-5928 |
| ispartof | Protein expression and purification, 1994-02, Vol.5 (1), p.37-43 |
| issn | 1046-5928 1096-0279 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_76449772 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Amino Acid Sequence Bacterial Proteins - biosynthesis Bacterial Proteins - isolation & purification Cellulose - analogs & derivatives Chromatography, Affinity Endodeoxyribonucleases - biosynthesis Endodeoxyribonucleases - isolation & purification Endoribonucleases - biosynthesis Endoribonucleases - isolation & purification Escherichia coli Protein Denaturation Protein Folding Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - isolation & purification Serratia marcescens - enzymology Serratia marcescens - genetics Urea |
| title | A Procedure for Renaturation and Purification of the Extracellular Serratia marcescens Nuclease from Genetically Engineered Escherichia coli |
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