Purification of the Cardiac Sarcoplasmic Reticulum Membrane Protein Phospholamban from Recombinant Escherichia Coli
Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S‐transferase (GST). GST–PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as...
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| Veröffentlicht in: | European journal of biochemistry 1997-09, Vol.248 (3), p.814-819 |
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| container_title | European journal of biochemistry |
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| creator | KrÖMer, Wolfgang J. Carafoli, Ernesto Bailey, James E. |
| description | Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S‐transferase (GST). GST–PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity‐purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1g cells we typically purified 13.5 nig fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification. |
| doi_str_mv | 10.1111/j.1432-1033.1997.00814.x |
| format | Article |
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GST–PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity‐purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1g cells we typically purified 13.5 nig fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.</description><subject>Animals</subject><subject>Blotting, Western</subject><subject>Calcium-Binding Proteins - chemistry</subject><subject>Calcium-Binding Proteins - genetics</subject><subject>Calcium-Binding Proteins - isolation & purification</subject><subject>cardiac</subject><subject>Dimerization</subject><subject>Dogs</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enteropeptidase - genetics</subject><subject>Enteropeptidase - metabolism</subject><subject>Escherichia coli - genetics</subject><subject>expression</subject><subject>Gene Expression</subject><subject>Glutathione Transferase - genetics</subject><subject>membrane protein</subject><subject>Myocardium - chemistry</subject><subject>Oligodeoxyribonucleotides - chemistry</subject><subject>phospholamban</subject><subject>Plasmids - genetics</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Sarcoplasmic Reticulum - chemistry</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkEtv1DAUhS0EKtOWn4DkFbuk14-JkwULGE1ppSJGtKwtx75RPIrjwU5E--_JMKOuuZtzpXPuQx8hlEHJlrrZl0wKXjAQomRNo0qAmsny-Q1ZvRpvyQqAyYI36-o9ucx5DwBVU6kLctEIybkQK5J3c_Kdt2bycaSxo1OPdGOS88bSR5NsPAwmB2_pT5y8nYc50O8Y2mRGpLsUJ_Qj3fUxH_o4mNCakXYphiVtY2j9aMaJbrPtMXnbe0M3cfDX5F1nhowfznpFft1unzZ3xcOPb_ebLw-FlUzKoganAFSFHQjnnJXcSrFGaCygrTpna1O5RVuF6JRUvKmxFp0UCMiV4uKKfDrtPaT4e8Y86eCzxWFYfo9z1qoRa66gWoL1KWhTzDlhpw_JB5NeNAN95K33-ohVH7HqI2_9j7d-XkY_nm_MbUD3OngGvPifT_4fP-DLf-_Vt9uvj0sn_gK4M5Cw</recordid><startdate>19970915</startdate><enddate>19970915</enddate><creator>KrÖMer, Wolfgang J.</creator><creator>Carafoli, Ernesto</creator><creator>Bailey, James E.</creator><general>Blackwell Science Ltd</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>19970915</creationdate><title>Purification of the Cardiac Sarcoplasmic Reticulum Membrane Protein Phospholamban from Recombinant Escherichia Coli</title><author>KrÖMer, Wolfgang J. ; Carafoli, Ernesto ; Bailey, James E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4144-80d70076ef03dddc42c435e09c0ec6fdc8a6d6fdb7eed747298e83f43e0e27723</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>Animals</topic><topic>Blotting, Western</topic><topic>Calcium-Binding Proteins - chemistry</topic><topic>Calcium-Binding Proteins - genetics</topic><topic>Calcium-Binding Proteins - isolation & purification</topic><topic>cardiac</topic><topic>Dimerization</topic><topic>Dogs</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enteropeptidase - genetics</topic><topic>Enteropeptidase - metabolism</topic><topic>Escherichia coli - genetics</topic><topic>expression</topic><topic>Gene Expression</topic><topic>Glutathione Transferase - genetics</topic><topic>membrane protein</topic><topic>Myocardium - chemistry</topic><topic>Oligodeoxyribonucleotides - chemistry</topic><topic>phospholamban</topic><topic>Plasmids - genetics</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Sarcoplasmic Reticulum - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>KrÖMer, Wolfgang J.</creatorcontrib><creatorcontrib>Carafoli, Ernesto</creatorcontrib><creatorcontrib>Bailey, James E.</creatorcontrib><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><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>KrÖMer, Wolfgang J.</au><au>Carafoli, Ernesto</au><au>Bailey, James E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification of the Cardiac Sarcoplasmic Reticulum Membrane Protein Phospholamban from Recombinant Escherichia Coli</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>1997-09-15</date><risdate>1997</risdate><volume>248</volume><issue>3</issue><spage>814</spage><epage>819</epage><pages>814-819</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><abstract>Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S‐transferase (GST). GST–PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity‐purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1g cells we typically purified 13.5 nig fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Ltd</pub><pmid>9342233</pmid><doi>10.1111/j.1432-1033.1997.00814.x</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | European journal of biochemistry, 1997-09, Vol.248 (3), p.814-819 |
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| language | eng |
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| source | Wiley Online Library - AutoHoldings Journals; MEDLINE; Alma/SFX Local Collection |
| subjects | Animals Blotting, Western Calcium-Binding Proteins - chemistry Calcium-Binding Proteins - genetics Calcium-Binding Proteins - isolation & purification cardiac Dimerization Dogs Electrophoresis, Polyacrylamide Gel Enteropeptidase - genetics Enteropeptidase - metabolism Escherichia coli - genetics expression Gene Expression Glutathione Transferase - genetics membrane protein Myocardium - chemistry Oligodeoxyribonucleotides - chemistry phospholamban Plasmids - genetics Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - isolation & purification Sarcoplasmic Reticulum - chemistry |
| title | Purification of the Cardiac Sarcoplasmic Reticulum Membrane Protein Phospholamban from Recombinant Escherichia Coli |
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