Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker
ABSTRACT In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoredu...
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| Veröffentlicht in: | Biomedical chromatography 2013-07, Vol.27 (7), p.859-865 |
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| creator | Pan, Yan Mak, Joon Wah Ong, Chin Eng |
| description | ABSTRACT
In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r2 = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10−2 μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km, Vmax and Ki) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd. |
| doi_str_mv | 10.1002/bmc.2872 |
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In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r2 = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10−2 μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km, Vmax and Ki) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd.</description><identifier>ISSN: 0269-3879</identifier><identifier>EISSN: 1099-0801</identifier><identifier>DOI: 10.1002/bmc.2872</identifier><identifier>PMID: 23386533</identifier><language>eng</language><publisher>England: Blackwell Publishing Ltd</publisher><subject>Aryl Hydrocarbon Hydroxylases - chemistry ; Aryl Hydrocarbon Hydroxylases - genetics ; Aryl Hydrocarbon Hydroxylases - metabolism ; Cell Membrane - metabolism ; Chromatography, High Pressure Liquid - methods ; Chromatography, Reverse-Phase - methods ; CYP2C19 ; Cytochrome P-450 CYP2C19 ; drug interaction ; Escherichia coli ; Escherichia coli - genetics ; HPLC ; Humans ; Immunoblotting ; in vitro ; Kinetics ; Limit of Detection ; Linear Models ; Microsomes, Liver - metabolism ; NADP - analysis ; NADP - metabolism ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Reproducibility of Results</subject><ispartof>Biomedical chromatography, 2013-07, Vol.27 (7), p.859-865</ispartof><rights>Copyright © 2013 John Wiley & Sons, Ltd.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4582-76bbd99d28e6b9b104f21819bd4f7d630682fd11c150f1cf8b7ca785c12ee9cf3</citedby><cites>FETCH-LOGICAL-c4582-76bbd99d28e6b9b104f21819bd4f7d630682fd11c150f1cf8b7ca785c12ee9cf3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fbmc.2872$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fbmc.2872$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27923,27924,45573,45574</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23386533$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pan, Yan</creatorcontrib><creatorcontrib>Mak, Joon Wah</creatorcontrib><creatorcontrib>Ong, Chin Eng</creatorcontrib><title>Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker</title><title>Biomedical chromatography</title><addtitle>Biomed. Chromatogr</addtitle><description>ABSTRACT
In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r2 = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10−2 μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km, Vmax and Ki) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd.</description><subject>Aryl Hydrocarbon Hydroxylases - chemistry</subject><subject>Aryl Hydrocarbon Hydroxylases - genetics</subject><subject>Aryl Hydrocarbon Hydroxylases - metabolism</subject><subject>Cell Membrane - metabolism</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Chromatography, Reverse-Phase - methods</subject><subject>CYP2C19</subject><subject>Cytochrome P-450 CYP2C19</subject><subject>drug interaction</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>HPLC</subject><subject>Humans</subject><subject>Immunoblotting</subject><subject>in vitro</subject><subject>Kinetics</subject><subject>Limit of Detection</subject><subject>Linear Models</subject><subject>Microsomes, Liver - metabolism</subject><subject>NADP - analysis</subject><subject>NADP - metabolism</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Reproducibility of Results</subject><issn>0269-3879</issn><issn>1099-0801</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1u1DAURi0EokNB4gmQl2WR4p_EsZcQlQ7StIxQEWJlOc41MSTx1M6UzlP0letRh7JCrO7m6Hz33g-h15ScUkLYu3a0p0zW7AlaUKJUQSShT9GCMKEKLmt1hF6k9JMQogSrn6MjxrkUFecLdLeEGWIYwo-wTRhuNxFS8mHCweF-O5oJ290cbB_DCHhdVgSfNN_XbzFrqMJ-wmfJ9hC97b3BNgwem6nDkGbTDj71I0zz3vRlXSzXqwaPMPehw3PACeINYJOwsbO_8fMOjyb-gvgSPXNmSPDqMI_R149nV82yWH0-_9S8XxW2rCQratG2nVIdkyBa1VJSOkYlVW1XuroTnAjJXEeppRVx1DrZ1tbUsrKUASjr-DE6efBuYrje5oX16JOFYTAT5E9oWnLFuKS8-j_KRU4jOfIvamNIKYLTm-jzYTtNid43pXNTet9URt8crNt2hO4R_FNNBooH4LcfYPdPkf5w0RyEB96nGW4f-fxVLWpeV_rb5bleCq6uLslFXvoezqSqyg</recordid><startdate>201307</startdate><enddate>201307</enddate><creator>Pan, Yan</creator><creator>Mak, Joon Wah</creator><creator>Ong, Chin Eng</creator><general>Blackwell Publishing Ltd</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>7QL</scope><scope>C1K</scope></search><sort><creationdate>201307</creationdate><title>Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker</title><author>Pan, Yan ; Mak, Joon Wah ; Ong, Chin Eng</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4582-76bbd99d28e6b9b104f21819bd4f7d630682fd11c150f1cf8b7ca785c12ee9cf3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Aryl Hydrocarbon Hydroxylases - chemistry</topic><topic>Aryl Hydrocarbon Hydroxylases - genetics</topic><topic>Aryl Hydrocarbon Hydroxylases - metabolism</topic><topic>Cell Membrane - metabolism</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Chromatography, Reverse-Phase - methods</topic><topic>CYP2C19</topic><topic>Cytochrome P-450 CYP2C19</topic><topic>drug interaction</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>HPLC</topic><topic>Humans</topic><topic>Immunoblotting</topic><topic>in vitro</topic><topic>Kinetics</topic><topic>Limit of Detection</topic><topic>Linear Models</topic><topic>Microsomes, Liver - metabolism</topic><topic>NADP - analysis</topic><topic>NADP - metabolism</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Reproducibility of Results</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pan, Yan</creatorcontrib><creatorcontrib>Mak, Joon Wah</creatorcontrib><creatorcontrib>Ong, Chin Eng</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>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><jtitle>Biomedical chromatography</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pan, Yan</au><au>Mak, Joon Wah</au><au>Ong, Chin Eng</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker</atitle><jtitle>Biomedical chromatography</jtitle><addtitle>Biomed. Chromatogr</addtitle><date>2013-07</date><risdate>2013</risdate><volume>27</volume><issue>7</issue><spage>859</spage><epage>865</epage><pages>859-865</pages><issn>0269-3879</issn><eissn>1099-0801</eissn><abstract>ABSTRACT
In this study, a simple and reliable reverse‐phase high‐performance liquid chromatography (RP‐HPLC) method was established and validated to analyze S‐mephenytoin 4‐hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co‐expressing CYP2C19 and NADPH‐CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP‐HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP‐HPLC assay showed good linearity (r2 = 1.00) with 4‐hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10−2 μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km, Vmax and Ki) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co‐expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP‐HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro. Copyright © 2013 John Wiley & Sons, Ltd.</abstract><cop>England</cop><pub>Blackwell Publishing Ltd</pub><pmid>23386533</pmid><doi>10.1002/bmc.2872</doi><tpages>7</tpages></addata></record> |
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| subjects | Aryl Hydrocarbon Hydroxylases - chemistry Aryl Hydrocarbon Hydroxylases - genetics Aryl Hydrocarbon Hydroxylases - metabolism Cell Membrane - metabolism Chromatography, High Pressure Liquid - methods Chromatography, Reverse-Phase - methods CYP2C19 Cytochrome P-450 CYP2C19 drug interaction Escherichia coli Escherichia coli - genetics HPLC Humans Immunoblotting in vitro Kinetics Limit of Detection Linear Models Microsomes, Liver - metabolism NADP - analysis NADP - metabolism Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Reproducibility of Results |
| title | Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker |
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