Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays
Spleen tyrosine kinase (Syk) is an important non‐receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high‐throughput assays, recombinant Syk protein is needed in bulk quantity...
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| Veröffentlicht in: | Biotechnology journal 2010-02, Vol.5 (2), p.201-212 |
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| creator | Singh, Deepika Rani, Reema Rajendran, Resmi Kaur, Namrata Jit Pandey, Abhinav Chopra, Puneet Jain, Tarun Jain, Manish Kumar Grover, Sonam Arya, Ranjana Saini, Kulvinder Singh |
| description | Spleen tyrosine kinase (Syk) is an important non‐receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high‐throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI‐2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze‐thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high‐throughput system. |
| doi_str_mv | 10.1002/biot.200900139 |
| format | Article |
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To identify small molecule inhibitors of Syk in high‐throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI‐2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze‐thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high‐throughput system.</description><identifier>ISSN: 1860-6768</identifier><identifier>EISSN: 1860-7314</identifier><identifier>DOI: 10.1002/biot.200900139</identifier><identifier>PMID: 20013945</identifier><language>eng</language><publisher>Weinheim: WILEY-VCH Verlag</publisher><subject>Baculoviridae - genetics ; Baculovirus ; Circular Dichroism ; Cloning, Molecular - methods ; Dictyostelium - enzymology ; Dictyostelium - genetics ; Dictyostelium - virology ; Dictyostelium discoideum ; Enzyme Stability ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Escherichia coli - virology ; Expression ; High-Throughput Screening Assays - methods ; Humans ; Intracellular Signaling Peptides and Proteins - chemistry ; Intracellular Signaling Peptides and Proteins - genetics ; Microscopy, Fluorescence ; Models, Molecular ; Phosphorylation ; Protein Structure, Secondary ; Protein-Tyrosine Kinases - biosynthesis ; Protein-Tyrosine Kinases - chemistry ; Protein-Tyrosine Kinases - genetics ; Purification ; Recombinant ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Syk ; Syk Kinase</subject><ispartof>Biotechnology journal, 2010-02, Vol.5 (2), p.201-212</ispartof><rights>Copyright © 2010 WILEY‐VCH Verlag GmbH & Co. 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To identify small molecule inhibitors of Syk in high‐throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI‐2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze‐thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high‐throughput system.</description><subject>Baculoviridae - genetics</subject><subject>Baculovirus</subject><subject>Circular Dichroism</subject><subject>Cloning, Molecular - methods</subject><subject>Dictyostelium - enzymology</subject><subject>Dictyostelium - genetics</subject><subject>Dictyostelium - virology</subject><subject>Dictyostelium discoideum</subject><subject>Enzyme Stability</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - virology</subject><subject>Expression</subject><subject>High-Throughput Screening Assays - methods</subject><subject>Humans</subject><subject>Intracellular Signaling Peptides and Proteins - chemistry</subject><subject>Intracellular Signaling Peptides and Proteins - genetics</subject><subject>Microscopy, Fluorescence</subject><subject>Models, Molecular</subject><subject>Phosphorylation</subject><subject>Protein Structure, Secondary</subject><subject>Protein-Tyrosine Kinases - biosynthesis</subject><subject>Protein-Tyrosine Kinases - chemistry</subject><subject>Protein-Tyrosine Kinases - genetics</subject><subject>Purification</subject><subject>Recombinant</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Syk</subject><subject>Syk Kinase</subject><issn>1860-6768</issn><issn>1860-7314</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE1P3DAQhi1UVCjtlSPyrXDIdhx_JUegFJAQVAK0FyTLcRzWbL6wE5X8-3q7y6o3TjMaPe-r0YPQIYEZAUh_FK4bZilADkBovoP2SSYgkZSwT5tdSJHtoS8hvAAwToF9RnvpP5rxffR0NTa6xaGvrW3xMPkuuNbipWt1sPj4flqeYG9N1xTx0g7YvvXehuC6mJnCYJuAq87jhXteJMPCd-Pzoh8HrEPQU_iKditdB_ttMw_Q46-Lh_Or5Obu8vr89CYxjLA8SakpmcziQ6URGa-kLjKiGdPGQMl5wfJSclsIUeVSCqtB5AVQkVaZqdKUEHqAvq97e9-9jjYMqnHB2LrWre3GoCTjMZkJ-jFJ4xcp8FXnbE2aqCR4W6neu0b7SRFQK_VqpV5t1cfA0aZ6LBpbbvF31xHI18AfV9vpgzp1dn338H95ss666Pxtm9V-qYSkkqv57aX6-RtycgtzNad_AWVUoB0</recordid><startdate>201002</startdate><enddate>201002</enddate><creator>Singh, Deepika</creator><creator>Rani, Reema</creator><creator>Rajendran, Resmi</creator><creator>Kaur, Namrata Jit</creator><creator>Pandey, Abhinav</creator><creator>Chopra, Puneet</creator><creator>Jain, Tarun</creator><creator>Jain, Manish Kumar</creator><creator>Grover, Sonam</creator><creator>Arya, Ranjana</creator><creator>Saini, Kulvinder Singh</creator><general>WILEY-VCH Verlag</general><general>WILEY‐VCH Verlag</general><scope>BSCLL</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>7X8</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>201002</creationdate><title>Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays</title><author>Singh, Deepika ; Rani, Reema ; Rajendran, Resmi ; Kaur, Namrata Jit ; Pandey, Abhinav ; Chopra, Puneet ; Jain, Tarun ; Jain, Manish Kumar ; Grover, Sonam ; Arya, Ranjana ; Saini, Kulvinder Singh</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4149-23cd478394dc685f7ab81a44acc0d55b49d75eb66f9776ea069b0362f8cf22113</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Baculoviridae - genetics</topic><topic>Baculovirus</topic><topic>Circular Dichroism</topic><topic>Cloning, Molecular - methods</topic><topic>Dictyostelium - enzymology</topic><topic>Dictyostelium - genetics</topic><topic>Dictyostelium - virology</topic><topic>Dictyostelium discoideum</topic><topic>Enzyme Stability</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - virology</topic><topic>Expression</topic><topic>High-Throughput Screening Assays - methods</topic><topic>Humans</topic><topic>Intracellular Signaling Peptides and Proteins - chemistry</topic><topic>Intracellular Signaling Peptides and Proteins - genetics</topic><topic>Microscopy, Fluorescence</topic><topic>Models, Molecular</topic><topic>Phosphorylation</topic><topic>Protein Structure, Secondary</topic><topic>Protein-Tyrosine Kinases - biosynthesis</topic><topic>Protein-Tyrosine Kinases - chemistry</topic><topic>Protein-Tyrosine Kinases - genetics</topic><topic>Purification</topic><topic>Recombinant</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Syk</topic><topic>Syk Kinase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Singh, Deepika</creatorcontrib><creatorcontrib>Rani, Reema</creatorcontrib><creatorcontrib>Rajendran, Resmi</creatorcontrib><creatorcontrib>Kaur, Namrata Jit</creatorcontrib><creatorcontrib>Pandey, Abhinav</creatorcontrib><creatorcontrib>Chopra, Puneet</creatorcontrib><creatorcontrib>Jain, Tarun</creatorcontrib><creatorcontrib>Jain, Manish Kumar</creatorcontrib><creatorcontrib>Grover, Sonam</creatorcontrib><creatorcontrib>Arya, Ranjana</creatorcontrib><creatorcontrib>Saini, Kulvinder Singh</creatorcontrib><collection>Istex</collection><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><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Biotechnology journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Singh, Deepika</au><au>Rani, Reema</au><au>Rajendran, Resmi</au><au>Kaur, Namrata Jit</au><au>Pandey, Abhinav</au><au>Chopra, Puneet</au><au>Jain, Tarun</au><au>Jain, Manish Kumar</au><au>Grover, Sonam</au><au>Arya, Ranjana</au><au>Saini, Kulvinder Singh</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays</atitle><jtitle>Biotechnology journal</jtitle><addtitle>Biotechnology Journal</addtitle><date>2010-02</date><risdate>2010</risdate><volume>5</volume><issue>2</issue><spage>201</spage><epage>212</epage><pages>201-212</pages><issn>1860-6768</issn><eissn>1860-7314</eissn><abstract>Spleen tyrosine kinase (Syk) is an important non‐receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high‐throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI‐2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze‐thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high‐throughput system.</abstract><cop>Weinheim</cop><pub>WILEY-VCH Verlag</pub><pmid>20013945</pmid><doi>10.1002/biot.200900139</doi><tpages>12</tpages></addata></record> |
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| subjects | Baculoviridae - genetics Baculovirus Circular Dichroism Cloning, Molecular - methods Dictyostelium - enzymology Dictyostelium - genetics Dictyostelium - virology Dictyostelium discoideum Enzyme Stability Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli - virology Expression High-Throughput Screening Assays - methods Humans Intracellular Signaling Peptides and Proteins - chemistry Intracellular Signaling Peptides and Proteins - genetics Microscopy, Fluorescence Models, Molecular Phosphorylation Protein Structure, Secondary Protein-Tyrosine Kinases - biosynthesis Protein-Tyrosine Kinases - chemistry Protein-Tyrosine Kinases - genetics Purification Recombinant Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - genetics Syk Syk Kinase |
| title | Human spleen tyrosine kinase (Syk) recombinant expression systems for high-throughput assays |
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