Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression
Purpose: To improve the stability and bioactivity of human basic fibroblast growth factor (hbFGF) by site-specific pegylation. Methods: Four new mutants of hbFGF were designed with substituted Asp68, Lys77, Glu78 and Arg81 with cysteine with the aid of bioinformatics technique, and then cloned into...
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| Veröffentlicht in: | Tropical journal of pharmaceutical research 2014-12, Vol.13 (10), p.1601 |
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| creator | Hadadian, Shahin Mirzahoseini, Hasan Shamassebi, Dariush Norouzian Shokrgozar, Mohamad Ali Bouzari, Saeid Asgari, Saeme |
| description | Purpose: To improve the stability and bioactivity of human basic
fibroblast growth factor (hbFGF) by site-specific pegylation. Methods:
Four new mutants of hbFGF were designed with substituted Asp68, Lys77,
Glu78 and Arg81 with cysteine with the aid of bioinformatics technique,
and then cloned into pET21a plasmid, transferred into E. coli BL21
(DE3). The expressed proteins were purified using cation exchange and
heparin affinity chromatography. Cysteine analogs of hbFGF were
PEGylated with 10 KDa PEG and purified using size exclusion
chromatography. Mitogenic activity and resistance against denaturation
agents were evaluated by MTT assay and fluorescence spectrophotometry,
respectively, and the results obtained were compared with the
non-PEGylated form. Results: Despite greater resistance against
denaturation agent (1.2 M guanidine hydrochloride for denaturation of
PEGylated mutants compared with 0.8 M for non-PEGylated forms), the
mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81
were retained at 79, 78.6, 83.3 and 75.6 %, respectively. Conclusion:
PEGylated hbFGF shows decreased mitogenic activity and increased
resistance against denaturation agent. |
| doi_str_mv | 10.4314/tjpr.v13i10.5 |
| format | Article |
| fullrecord | <record><control><sourceid>bioline_cross</sourceid><recordid>TN_cdi_crossref_primary_10_4314_tjpr_v13i10_5</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>cria_bioline_pr_pr14220</sourcerecordid><originalsourceid>FETCH-LOGICAL-b315t-7431e97e4cbdd319c35648ad8f242303256fc3e7799ea36a245b8849a10fc3673</originalsourceid><addsrcrecordid>eNpFkM9LwzAcxYMoOKdH7znqoTM_2-Y469oJAz3oOaRpumV0zUiyuf33dmwofOF9-fB48B4AjxhNGMXsJa63frLH1A6AX4ER5iJNRE6y68vPhUhvwV0Ia4R4KgQegV2xMhsXTGd0tHsDP2fVsVPRuh66FhbHEI3tDZz2qnPLcGLz3Ub18FUFq2Fpa-_qToUIK-9-4gqWSkfn4dOqLqvyGSbwzQS77KHqGzg7bL0JYci-Bzet6oJ5uOgYfJezr2KeLD6q92K6SGqKeUyyoZYRmWG6bhqKhaY8Zblq8pYwQhElPG01NVkmhFE0VYTxOs-ZUBgNPM3oGCTnXO1dCN60cuvtRvmjxEieNpOnzeR5M8kH_-Tsr63rht5_du2tkv9wOMwIQfQXsl1x2A</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype></control><display><type>article</type><title>Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression</title><source>DOAJ Directory of Open Access Journals</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>African Journals Online (Open Access)</source><source>Bioline International</source><source>Free Full-Text Journals in Chemistry</source><creator>Hadadian, Shahin ; Mirzahoseini, Hasan ; Shamassebi, Dariush Norouzian ; Shokrgozar, Mohamad Ali ; Bouzari, Saeid ; Asgari, Saeme</creator><creatorcontrib>Hadadian, Shahin ; Mirzahoseini, Hasan ; Shamassebi, Dariush Norouzian ; Shokrgozar, Mohamad Ali ; Bouzari, Saeid ; Asgari, Saeme</creatorcontrib><description>Purpose: To improve the stability and bioactivity of human basic
fibroblast growth factor (hbFGF) by site-specific pegylation. Methods:
Four new mutants of hbFGF were designed with substituted Asp68, Lys77,
Glu78 and Arg81 with cysteine with the aid of bioinformatics technique,
and then cloned into pET21a plasmid, transferred into E. coli BL21
(DE3). The expressed proteins were purified using cation exchange and
heparin affinity chromatography. Cysteine analogs of hbFGF were
PEGylated with 10 KDa PEG and purified using size exclusion
chromatography. Mitogenic activity and resistance against denaturation
agents were evaluated by MTT assay and fluorescence spectrophotometry,
respectively, and the results obtained were compared with the
non-PEGylated form. Results: Despite greater resistance against
denaturation agent (1.2 M guanidine hydrochloride for denaturation of
PEGylated mutants compared with 0.8 M for non-PEGylated forms), the
mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81
were retained at 79, 78.6, 83.3 and 75.6 %, respectively. Conclusion:
PEGylated hbFGF shows decreased mitogenic activity and increased
resistance against denaturation agent.</description><identifier>ISSN: 1596-5996</identifier><identifier>EISSN: 1596-9827</identifier><identifier>DOI: 10.4314/tjpr.v13i10.5</identifier><language>eng</language><publisher>Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria</publisher><subject>Bioinformatics ; Cysteine analog ; Denaturation agent ; Fibroblast growth factor ; Guanidine hydrochloride ; Mitogenic activity ; PEGylation</subject><ispartof>Tropical journal of pharmaceutical research, 2014-12, Vol.13 (10), p.1601</ispartof><rights>Copyright 2014 - Tropical Journal of Pharmaceutical Research</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-b315t-7431e97e4cbdd319c35648ad8f242303256fc3e7799ea36a245b8849a10fc3673</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785,865,27926,27927,79428</link.rule.ids></links><search><creatorcontrib>Hadadian, Shahin</creatorcontrib><creatorcontrib>Mirzahoseini, Hasan</creatorcontrib><creatorcontrib>Shamassebi, Dariush Norouzian</creatorcontrib><creatorcontrib>Shokrgozar, Mohamad Ali</creatorcontrib><creatorcontrib>Bouzari, Saeid</creatorcontrib><creatorcontrib>Asgari, Saeme</creatorcontrib><title>Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression</title><title>Tropical journal of pharmaceutical research</title><description>Purpose: To improve the stability and bioactivity of human basic
fibroblast growth factor (hbFGF) by site-specific pegylation. Methods:
Four new mutants of hbFGF were designed with substituted Asp68, Lys77,
Glu78 and Arg81 with cysteine with the aid of bioinformatics technique,
and then cloned into pET21a plasmid, transferred into E. coli BL21
(DE3). The expressed proteins were purified using cation exchange and
heparin affinity chromatography. Cysteine analogs of hbFGF were
PEGylated with 10 KDa PEG and purified using size exclusion
chromatography. Mitogenic activity and resistance against denaturation
agents were evaluated by MTT assay and fluorescence spectrophotometry,
respectively, and the results obtained were compared with the
non-PEGylated form. Results: Despite greater resistance against
denaturation agent (1.2 M guanidine hydrochloride for denaturation of
PEGylated mutants compared with 0.8 M for non-PEGylated forms), the
mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81
were retained at 79, 78.6, 83.3 and 75.6 %, respectively. Conclusion:
PEGylated hbFGF shows decreased mitogenic activity and increased
resistance against denaturation agent.</description><subject>Bioinformatics</subject><subject>Cysteine analog</subject><subject>Denaturation agent</subject><subject>Fibroblast growth factor</subject><subject>Guanidine hydrochloride</subject><subject>Mitogenic activity</subject><subject>PEGylation</subject><issn>1596-5996</issn><issn>1596-9827</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>RBI</sourceid><recordid>eNpFkM9LwzAcxYMoOKdH7znqoTM_2-Y469oJAz3oOaRpumV0zUiyuf33dmwofOF9-fB48B4AjxhNGMXsJa63frLH1A6AX4ER5iJNRE6y68vPhUhvwV0Ia4R4KgQegV2xMhsXTGd0tHsDP2fVsVPRuh66FhbHEI3tDZz2qnPLcGLz3Ub18FUFq2Fpa-_qToUIK-9-4gqWSkfn4dOqLqvyGSbwzQS77KHqGzg7bL0JYci-Bzet6oJ5uOgYfJezr2KeLD6q92K6SGqKeUyyoZYRmWG6bhqKhaY8Zblq8pYwQhElPG01NVkmhFE0VYTxOs-ZUBgNPM3oGCTnXO1dCN60cuvtRvmjxEieNpOnzeR5M8kH_-Tsr63rht5_du2tkv9wOMwIQfQXsl1x2A</recordid><startdate>20141209</startdate><enddate>20141209</enddate><creator>Hadadian, Shahin</creator><creator>Mirzahoseini, Hasan</creator><creator>Shamassebi, Dariush Norouzian</creator><creator>Shokrgozar, Mohamad Ali</creator><creator>Bouzari, Saeid</creator><creator>Asgari, Saeme</creator><general>Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria</general><scope>RBI</scope><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>20141209</creationdate><title>Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression</title><author>Hadadian, Shahin ; Mirzahoseini, Hasan ; Shamassebi, Dariush Norouzian ; Shokrgozar, Mohamad Ali ; Bouzari, Saeid ; Asgari, Saeme</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-b315t-7431e97e4cbdd319c35648ad8f242303256fc3e7799ea36a245b8849a10fc3673</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Bioinformatics</topic><topic>Cysteine analog</topic><topic>Denaturation agent</topic><topic>Fibroblast growth factor</topic><topic>Guanidine hydrochloride</topic><topic>Mitogenic activity</topic><topic>PEGylation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hadadian, Shahin</creatorcontrib><creatorcontrib>Mirzahoseini, Hasan</creatorcontrib><creatorcontrib>Shamassebi, Dariush Norouzian</creatorcontrib><creatorcontrib>Shokrgozar, Mohamad Ali</creatorcontrib><creatorcontrib>Bouzari, Saeid</creatorcontrib><creatorcontrib>Asgari, Saeme</creatorcontrib><collection>Bioline International</collection><collection>CrossRef</collection><jtitle>Tropical journal of pharmaceutical research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hadadian, Shahin</au><au>Mirzahoseini, Hasan</au><au>Shamassebi, Dariush Norouzian</au><au>Shokrgozar, Mohamad Ali</au><au>Bouzari, Saeid</au><au>Asgari, Saeme</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression</atitle><jtitle>Tropical journal of pharmaceutical research</jtitle><date>2014-12-09</date><risdate>2014</risdate><volume>13</volume><issue>10</issue><spage>1601</spage><pages>1601-</pages><issn>1596-5996</issn><eissn>1596-9827</eissn><abstract>Purpose: To improve the stability and bioactivity of human basic
fibroblast growth factor (hbFGF) by site-specific pegylation. Methods:
Four new mutants of hbFGF were designed with substituted Asp68, Lys77,
Glu78 and Arg81 with cysteine with the aid of bioinformatics technique,
and then cloned into pET21a plasmid, transferred into E. coli BL21
(DE3). The expressed proteins were purified using cation exchange and
heparin affinity chromatography. Cysteine analogs of hbFGF were
PEGylated with 10 KDa PEG and purified using size exclusion
chromatography. Mitogenic activity and resistance against denaturation
agents were evaluated by MTT assay and fluorescence spectrophotometry,
respectively, and the results obtained were compared with the
non-PEGylated form. Results: Despite greater resistance against
denaturation agent (1.2 M guanidine hydrochloride for denaturation of
PEGylated mutants compared with 0.8 M for non-PEGylated forms), the
mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81
were retained at 79, 78.6, 83.3 and 75.6 %, respectively. Conclusion:
PEGylated hbFGF shows decreased mitogenic activity and increased
resistance against denaturation agent.</abstract><pub>Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria</pub><doi>10.4314/tjpr.v13i10.5</doi><oa>free_for_read</oa></addata></record> |
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| source | DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; African Journals Online (Open Access); Bioline International; Free Full-Text Journals in Chemistry |
| subjects | Bioinformatics Cysteine analog Denaturation agent Fibroblast growth factor Guanidine hydrochloride Mitogenic activity PEGylation |
| title | Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression |
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