Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification
The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important feature...
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| Veröffentlicht in: | Protein expression and purification 2016-03, Vol.119, p.102-109 |
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| creator | Zieliński, Marcin Wójtowicz-Krawiec, Anna Mikiewicz, Diana Kęsik-Brodacka, Małgorzata Cecuda-Adamczewska, Violetta Marciniak-Rusek, Alina Sokołowska, Iwona Łukasiewicz, Natalia Gurba, Lidia Odrowąż-Sypniewski, Michał Baran, Piotr Płucienniczak, Grażyna Płucienniczak, Andrzej Borowicz, Piotr Szewczyk, Bogusław |
| description | The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5–7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P |
| doi_str_mv | 10.1016/j.pep.2015.11.017 |
| format | Article |
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•An impressive yield of soluble PAM per liter of media.•Glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue as a novel PAM substrate.•GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care.•This work provides a valuable alternative method for preparing bioactivity peptides.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/j.pep.2015.11.017</identifier><identifier>PMID: 26614892</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amidine-Lyases - biosynthesis ; Amidine-Lyases - chemistry ; Amidine-Lyases - isolation & purification ; Animals ; Blood Glucose ; Chinese hamster ovary cell ; CHO Cells ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Cricetinae ; Cricetulus ; Diabetes Mellitus, Experimental - drug therapy ; Dihydrofolate reductase ; Drug Evaluation, Preclinical ; Female ; Human peptidylglycine α-amidating monooxygenase ; Hypoglycemic activity ; Hypoglycemic Agents - chemistry ; Hypoglycemic Agents - pharmacology ; Insulin - analogs & derivatives ; Insulin - chemistry ; Insulin - pharmacology ; Insulin amide ; Male ; Methotrexate ; Mixed Function Oxygenases - biosynthesis ; Mixed Function Oxygenases - chemistry ; Mixed Function Oxygenases - isolation & purification ; Rats, Wistar ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - isolation & purification</subject><ispartof>Protein expression and purification, 2016-03, Vol.119, p.102-109</ispartof><rights>2015 Elsevier Inc.</rights><rights>Copyright © 2015 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-a58f4b0d9ef4e62c430ea44b5a82f887a9f9bd6e926c3aa81ae53ae16e8237443</citedby><cites>FETCH-LOGICAL-c386t-a58f4b0d9ef4e62c430ea44b5a82f887a9f9bd6e926c3aa81ae53ae16e8237443</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.pep.2015.11.017$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26614892$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zieliński, Marcin</creatorcontrib><creatorcontrib>Wójtowicz-Krawiec, Anna</creatorcontrib><creatorcontrib>Mikiewicz, Diana</creatorcontrib><creatorcontrib>Kęsik-Brodacka, Małgorzata</creatorcontrib><creatorcontrib>Cecuda-Adamczewska, Violetta</creatorcontrib><creatorcontrib>Marciniak-Rusek, Alina</creatorcontrib><creatorcontrib>Sokołowska, Iwona</creatorcontrib><creatorcontrib>Łukasiewicz, Natalia</creatorcontrib><creatorcontrib>Gurba, Lidia</creatorcontrib><creatorcontrib>Odrowąż-Sypniewski, Michał</creatorcontrib><creatorcontrib>Baran, Piotr</creatorcontrib><creatorcontrib>Płucienniczak, Grażyna</creatorcontrib><creatorcontrib>Płucienniczak, Andrzej</creatorcontrib><creatorcontrib>Borowicz, Piotr</creatorcontrib><creatorcontrib>Szewczyk, Bogusław</creatorcontrib><title>Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5–7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5–2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.
•An impressive yield of soluble PAM per liter of media.•Glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue as a novel PAM substrate.•GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care.•This work provides a valuable alternative method for preparing bioactivity peptides.</description><subject>Amidine-Lyases - biosynthesis</subject><subject>Amidine-Lyases - chemistry</subject><subject>Amidine-Lyases - isolation & purification</subject><subject>Animals</subject><subject>Blood Glucose</subject><subject>Chinese hamster ovary cell</subject><subject>CHO Cells</subject><subject>Chromatography, Gel</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Diabetes Mellitus, Experimental - drug therapy</subject><subject>Dihydrofolate reductase</subject><subject>Drug Evaluation, Preclinical</subject><subject>Female</subject><subject>Human peptidylglycine α-amidating monooxygenase</subject><subject>Hypoglycemic activity</subject><subject>Hypoglycemic Agents - chemistry</subject><subject>Hypoglycemic Agents - pharmacology</subject><subject>Insulin - analogs & derivatives</subject><subject>Insulin - chemistry</subject><subject>Insulin - pharmacology</subject><subject>Insulin amide</subject><subject>Male</subject><subject>Methotrexate</subject><subject>Mixed Function Oxygenases - biosynthesis</subject><subject>Mixed Function Oxygenases - chemistry</subject><subject>Mixed Function Oxygenases - isolation & purification</subject><subject>Rats, Wistar</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - isolation & purification</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1TAQRSMEoqXlA9ggL9kk2I7jOGKFnkqLVKkburYm9vjhp8QOcYL6fqN_wo_wTXX0CktY2dKce2d0b1G8Y7RilMmPh2rCqeKUNRVjFWXti-Kc0U6WlLfdy-0vZNl0XJ0Vb1I6UMqYpM3r4oxLyYTq-HnxePUwzZiSj4FER2Y0cex9gLCQ7-sIgfTercEseQ4DyesWb4_DfjgaH5D8_lXC6C0sPuzJGEOMD8c9BkhIfCC7mzticBgSgWCJXxJZ88DFOQ_TOmQCsmncr5i11jtvYNtzWbxyMCR8-_xeFPdfrr7tbsrbu-uvu8-3pamVXEpolBM9tR06gZIbUVMEIfoGFHdKtdC5rrcSOy5NDaAYYFMDMomK160Q9UXx4eQ7zfHHimnRo0_bvRAwrkkz1XLFec7v_2grqVJ1rWhG2Qk1c0xpRqen2Y8wHzWjemtNH3SOUW-tacZ0bi1r3j_br_2I9q_iT00Z-HQCMOfx0-Osk_EYDFqfG1u0jf4f9k_29qwk</recordid><startdate>201603</startdate><enddate>201603</enddate><creator>Zieliński, Marcin</creator><creator>Wójtowicz-Krawiec, Anna</creator><creator>Mikiewicz, Diana</creator><creator>Kęsik-Brodacka, Małgorzata</creator><creator>Cecuda-Adamczewska, Violetta</creator><creator>Marciniak-Rusek, Alina</creator><creator>Sokołowska, Iwona</creator><creator>Łukasiewicz, Natalia</creator><creator>Gurba, Lidia</creator><creator>Odrowąż-Sypniewski, Michał</creator><creator>Baran, Piotr</creator><creator>Płucienniczak, Grażyna</creator><creator>Płucienniczak, Andrzej</creator><creator>Borowicz, Piotr</creator><creator>Szewczyk, Bogusław</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><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>201603</creationdate><title>Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification</title><author>Zieliński, Marcin ; Wójtowicz-Krawiec, Anna ; Mikiewicz, Diana ; Kęsik-Brodacka, Małgorzata ; Cecuda-Adamczewska, Violetta ; Marciniak-Rusek, Alina ; Sokołowska, Iwona ; Łukasiewicz, Natalia ; Gurba, Lidia ; Odrowąż-Sypniewski, Michał ; Baran, Piotr ; Płucienniczak, Grażyna ; Płucienniczak, Andrzej ; Borowicz, Piotr ; Szewczyk, Bogusław</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-a58f4b0d9ef4e62c430ea44b5a82f887a9f9bd6e926c3aa81ae53ae16e8237443</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Amidine-Lyases - biosynthesis</topic><topic>Amidine-Lyases - chemistry</topic><topic>Amidine-Lyases - isolation & purification</topic><topic>Animals</topic><topic>Blood Glucose</topic><topic>Chinese hamster ovary cell</topic><topic>CHO Cells</topic><topic>Chromatography, Gel</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Diabetes Mellitus, Experimental - drug therapy</topic><topic>Dihydrofolate reductase</topic><topic>Drug Evaluation, Preclinical</topic><topic>Female</topic><topic>Human peptidylglycine α-amidating monooxygenase</topic><topic>Hypoglycemic activity</topic><topic>Hypoglycemic Agents - chemistry</topic><topic>Hypoglycemic Agents - pharmacology</topic><topic>Insulin - analogs & derivatives</topic><topic>Insulin - chemistry</topic><topic>Insulin - pharmacology</topic><topic>Insulin amide</topic><topic>Male</topic><topic>Methotrexate</topic><topic>Mixed Function Oxygenases - biosynthesis</topic><topic>Mixed Function Oxygenases - chemistry</topic><topic>Mixed Function Oxygenases - isolation & purification</topic><topic>Rats, Wistar</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - isolation & purification</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zieliński, Marcin</creatorcontrib><creatorcontrib>Wójtowicz-Krawiec, Anna</creatorcontrib><creatorcontrib>Mikiewicz, Diana</creatorcontrib><creatorcontrib>Kęsik-Brodacka, Małgorzata</creatorcontrib><creatorcontrib>Cecuda-Adamczewska, Violetta</creatorcontrib><creatorcontrib>Marciniak-Rusek, Alina</creatorcontrib><creatorcontrib>Sokołowska, Iwona</creatorcontrib><creatorcontrib>Łukasiewicz, Natalia</creatorcontrib><creatorcontrib>Gurba, Lidia</creatorcontrib><creatorcontrib>Odrowąż-Sypniewski, Michał</creatorcontrib><creatorcontrib>Baran, Piotr</creatorcontrib><creatorcontrib>Płucienniczak, Grażyna</creatorcontrib><creatorcontrib>Płucienniczak, Andrzej</creatorcontrib><creatorcontrib>Borowicz, Piotr</creatorcontrib><creatorcontrib>Szewczyk, Bogusław</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><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zieliński, Marcin</au><au>Wójtowicz-Krawiec, Anna</au><au>Mikiewicz, Diana</au><au>Kęsik-Brodacka, Małgorzata</au><au>Cecuda-Adamczewska, Violetta</au><au>Marciniak-Rusek, Alina</au><au>Sokołowska, Iwona</au><au>Łukasiewicz, Natalia</au><au>Gurba, Lidia</au><au>Odrowąż-Sypniewski, Michał</au><au>Baran, Piotr</au><au>Płucienniczak, Grażyna</au><au>Płucienniczak, Andrzej</au><au>Borowicz, Piotr</au><au>Szewczyk, Bogusław</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2016-03</date><risdate>2016</risdate><volume>119</volume><spage>102</spage><epage>109</epage><pages>102-109</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5–7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5–2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.
•An impressive yield of soluble PAM per liter of media.•Glycine-extended A22(G)-B31(K)–B32(R) human insulin analogue as a novel PAM substrate.•GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care.•This work provides a valuable alternative method for preparing bioactivity peptides.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>26614892</pmid><doi>10.1016/j.pep.2015.11.017</doi><tpages>8</tpages></addata></record> |
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| ispartof | Protein expression and purification, 2016-03, Vol.119, p.102-109 |
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| subjects | Amidine-Lyases - biosynthesis Amidine-Lyases - chemistry Amidine-Lyases - isolation & purification Animals Blood Glucose Chinese hamster ovary cell CHO Cells Chromatography, Gel Chromatography, High Pressure Liquid Cricetinae Cricetulus Diabetes Mellitus, Experimental - drug therapy Dihydrofolate reductase Drug Evaluation, Preclinical Female Human peptidylglycine α-amidating monooxygenase Hypoglycemic activity Hypoglycemic Agents - chemistry Hypoglycemic Agents - pharmacology Insulin - analogs & derivatives Insulin - chemistry Insulin - pharmacology Insulin amide Male Methotrexate Mixed Function Oxygenases - biosynthesis Mixed Function Oxygenases - chemistry Mixed Function Oxygenases - isolation & purification Rats, Wistar Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - isolation & purification |
| title | Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification |
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