Expression and Characterization of Recombinant Ecarin
The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to...
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| Veröffentlicht in: | Protein Journal 2012-06, Vol.31 (5), p.353-358 |
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| creator | Jonebring, Anna Lange, Ute Bucha, Elke Deinum, Johanna Elg, Margareta Lövgren, Ann |
| description | The snake venom protease ecarin from
Echis carinatus
was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a
K
m
value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the
K
m
for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin. |
| doi_str_mv | 10.1007/s10930-012-9409-6 |
| format | Article |
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Echis carinatus
was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a
K
m
value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the
K
m
for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.</description><identifier>ISSN: 1572-3887</identifier><identifier>EISSN: 1573-4943</identifier><identifier>EISSN: 1875-8355</identifier><identifier>DOI: 10.1007/s10930-012-9409-6</identifier><identifier>PMID: 22528138</identifier><language>eng</language><publisher>Boston: Springer US</publisher><subject>Animal Anatomy ; Animals ; Biochemistry ; Bioorganic Chemistry ; Cell culture ; Chemistry ; Chemistry and Materials Science ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Echis carinatus ; Endopeptidases - genetics ; Endopeptidases - metabolism ; Enzyme Activation ; Fibrinolytic Agents - metabolism ; Gene Expression ; Histology ; Humans ; Kinetics ; Morphology ; Organic Chemistry ; Proteases ; Proteinase ; Proteins ; Prothrombin ; Prothrombin - metabolism ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Snakes ; Thrombin ; Venom ; Viper Venoms - genetics ; Viper Venoms - metabolism ; Viperidae - genetics ; Viperidae - metabolism</subject><ispartof>Protein Journal, 2012-06, Vol.31 (5), p.353-358</ispartof><rights>The Author(s) 2012</rights><rights>COPYRIGHT 2012 Springer</rights><rights>Springer Science+Business Media, LLC 2012</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c570t-c38ad19a9474fbd8288ad8ae08386d69a0b50762817fd37fd9552cc4439f16443</citedby><cites>FETCH-LOGICAL-c570t-c38ad19a9474fbd8288ad8ae08386d69a0b50762817fd37fd9552cc4439f16443</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10930-012-9409-6$$EPDF$$P50$$Gspringer$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10930-012-9409-6$$EHTML$$P50$$Gspringer$$Hfree_for_read</linktohtml><link.rule.ids>230,315,782,786,887,27931,27932,41495,42564,51326</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22528138$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jonebring, Anna</creatorcontrib><creatorcontrib>Lange, Ute</creatorcontrib><creatorcontrib>Bucha, Elke</creatorcontrib><creatorcontrib>Deinum, Johanna</creatorcontrib><creatorcontrib>Elg, Margareta</creatorcontrib><creatorcontrib>Lövgren, Ann</creatorcontrib><title>Expression and Characterization of Recombinant Ecarin</title><title>Protein Journal</title><addtitle>Protein J</addtitle><addtitle>Protein J</addtitle><description>The snake venom protease ecarin from
Echis carinatus
was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a
K
m
value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the
K
m
for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.</description><subject>Animal Anatomy</subject><subject>Animals</subject><subject>Biochemistry</subject><subject>Bioorganic Chemistry</subject><subject>Cell culture</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>CHO Cells</subject><subject>Cloning, Molecular</subject><subject>Cricetinae</subject><subject>Echis carinatus</subject><subject>Endopeptidases - genetics</subject><subject>Endopeptidases - metabolism</subject><subject>Enzyme Activation</subject><subject>Fibrinolytic Agents - metabolism</subject><subject>Gene Expression</subject><subject>Histology</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Morphology</subject><subject>Organic Chemistry</subject><subject>Proteases</subject><subject>Proteinase</subject><subject>Proteins</subject><subject>Prothrombin</subject><subject>Prothrombin - metabolism</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Snakes</subject><subject>Thrombin</subject><subject>Venom</subject><subject>Viper Venoms - genetics</subject><subject>Viper Venoms - metabolism</subject><subject>Viperidae - genetics</subject><subject>Viperidae - metabolism</subject><issn>1572-3887</issn><issn>1573-4943</issn><issn>1875-8355</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqNkl1rFDEUhoMotlZ_gDey4I03U0--JsmNUJZtFQqC6HXIZjLblJlkTWZF_fWe2V1LKwoSwsnHc17OObyEvKRwTgHU20rBcGiAssYIME37iJxSqXgjjOCP92fWcK3VCXlW6y0A00axp-SEMck05fqUyNX3bQm1xpwWLnWL5Y0rzk-hxJ9umh9zv_gUfB7XMbk0LVbelZiekye9G2p4cYxn5Mvl6vPyfXP98erD8uK68VLB1HiuXUeNM0KJft1ppvGuXQDNddu1xsFagmqxFNV3HLeRknkvBDc9bTGckXcH3e1uPYbOhzQVN9htiaMrP2x20T78SfHGbvI3y7kG7BEF3hwFSv66C3WyY6w-DINLIe-qnQcosDqA_0AZNVpwYRB9_Qd6m3cl4ST2FCrSffFHauOGYGPqM5boZ1F7oWgrJYDgSJ3_hcLVhTH6nEIf8f1BAj0k-JJrLaG_GwcFO9vCHmxh0RZ2toVtMefV_TneZfz2AQLsAFT8SptQ7nf0L9VfZt-_Vg</recordid><startdate>20120601</startdate><enddate>20120601</enddate><creator>Jonebring, Anna</creator><creator>Lange, Ute</creator><creator>Bucha, Elke</creator><creator>Deinum, Johanna</creator><creator>Elg, Margareta</creator><creator>Lövgren, Ann</creator><general>Springer US</general><general>Springer</general><general>Springer Nature B.V</general><scope>C6C</scope><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>3V.</scope><scope>7QL</scope><scope>7TK</scope><scope>7TM</scope><scope>7U7</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PDBOC</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20120601</creationdate><title>Expression and Characterization of Recombinant Ecarin</title><author>Jonebring, Anna ; Lange, Ute ; Bucha, Elke ; Deinum, Johanna ; Elg, Margareta ; Lövgren, Ann</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c570t-c38ad19a9474fbd8288ad8ae08386d69a0b50762817fd37fd9552cc4439f16443</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animal Anatomy</topic><topic>Animals</topic><topic>Biochemistry</topic><topic>Bioorganic Chemistry</topic><topic>Cell culture</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>CHO Cells</topic><topic>Cloning, Molecular</topic><topic>Cricetinae</topic><topic>Echis carinatus</topic><topic>Endopeptidases - genetics</topic><topic>Endopeptidases - metabolism</topic><topic>Enzyme Activation</topic><topic>Fibrinolytic Agents - metabolism</topic><topic>Gene Expression</topic><topic>Histology</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Morphology</topic><topic>Organic Chemistry</topic><topic>Proteases</topic><topic>Proteinase</topic><topic>Proteins</topic><topic>Prothrombin</topic><topic>Prothrombin - metabolism</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Snakes</topic><topic>Thrombin</topic><topic>Venom</topic><topic>Viper Venoms - genetics</topic><topic>Viper Venoms - metabolism</topic><topic>Viperidae - genetics</topic><topic>Viperidae - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jonebring, Anna</creatorcontrib><creatorcontrib>Lange, Ute</creatorcontrib><creatorcontrib>Bucha, Elke</creatorcontrib><creatorcontrib>Deinum, Johanna</creatorcontrib><creatorcontrib>Elg, Margareta</creatorcontrib><creatorcontrib>Lövgren, Ann</creatorcontrib><collection>Springer Nature OA/Free Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>Proquest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Materials Science Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Protein Journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jonebring, Anna</au><au>Lange, Ute</au><au>Bucha, Elke</au><au>Deinum, Johanna</au><au>Elg, Margareta</au><au>Lövgren, Ann</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression and Characterization of Recombinant Ecarin</atitle><jtitle>Protein Journal</jtitle><stitle>Protein J</stitle><addtitle>Protein J</addtitle><date>2012-06-01</date><risdate>2012</risdate><volume>31</volume><issue>5</issue><spage>353</spage><epage>358</epage><pages>353-358</pages><issn>1572-3887</issn><eissn>1573-4943</eissn><eissn>1875-8355</eissn><abstract>The snake venom protease ecarin from
Echis carinatus
was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a
K
m
value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the
K
m
for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.</abstract><cop>Boston</cop><pub>Springer US</pub><pmid>22528138</pmid><doi>10.1007/s10930-012-9409-6</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1572-3887 |
| ispartof | Protein Journal, 2012-06, Vol.31 (5), p.353-358 |
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| language | eng |
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| source | MEDLINE; SpringerNature Journals |
| subjects | Animal Anatomy Animals Biochemistry Bioorganic Chemistry Cell culture Chemistry Chemistry and Materials Science CHO Cells Cloning, Molecular Cricetinae Echis carinatus Endopeptidases - genetics Endopeptidases - metabolism Enzyme Activation Fibrinolytic Agents - metabolism Gene Expression Histology Humans Kinetics Morphology Organic Chemistry Proteases Proteinase Proteins Prothrombin Prothrombin - metabolism Recombinant Proteins - genetics Recombinant Proteins - metabolism Snakes Thrombin Venom Viper Venoms - genetics Viper Venoms - metabolism Viperidae - genetics Viperidae - metabolism |
| title | Expression and Characterization of Recombinant Ecarin |
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