One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis
Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a...
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| Veröffentlicht in: | PloS one 2013-04, Vol.8 (4), p.e60786-e60786 |
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| creator | Shih, Kuo-Shun Lin, Chih-Chang Hung, Hsiao-Fang Yang, Yu-Chi Wang, Chung-An Jeng, Kee-Ching Fu, Hua-Wen |
| description | Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection. |
| doi_str_mv | 10.1371/journal.pone.0060786 |
| format | Article |
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HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0060786</identifier><identifier>PMID: 23577158</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Anion exchanging ; Anion-exchange chromatography ; Antigens ; Aquaporins ; Asthma ; Bacillus subtilis ; Bacillus subtilis - genetics ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - isolation & purification ; Bacterial Proteins - pharmacology ; Biology ; Bladder ; Bladder cancer ; Cancer ; Care and treatment ; Cellular biology ; Chemical compounds ; Chemistry ; Chromatography ; Chromatography, Ion Exchange - methods ; Columns (structural) ; Cytokines ; Dextrans ; Diagnosis ; Diagnostic reagents ; Diagnostic systems ; Drug development ; Drugs ; E coli ; Escherichia coli ; Gastric cancer ; Gene Expression ; Health aspects ; Helicobacter infections ; Helicobacter pylori ; Humans ; Impurities ; Infection ; Infections ; Inflammation ; Leukocytes (neutrophilic) ; Medicine ; Neutrophils ; Neutrophils - drug effects ; Neutrophils - metabolism ; Oxygen ; pH effects ; Pharmacology ; Protein binding ; Protein purification ; Protein structure ; Proteins ; Purification ; Purity ; Reactive oxygen species ; Reactive Oxygen Species - metabolism ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - pharmacology ; Resins ; Secondary structure ; Stomach cancer ; Tumor necrosis factor-TNF ; Ulcers ; Urinary bladder ; Vaccines ; Virulence ; Virulence factors</subject><ispartof>PloS one, 2013-04, Vol.8 (4), p.e60786-e60786</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Shih et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Shih et al 2013 Shih et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-45450e5d7e10761e797588ab63044780ff950c5358f042433c29067be82fb2c33</citedby><cites>FETCH-LOGICAL-c692t-45450e5d7e10761e797588ab63044780ff950c5358f042433c29067be82fb2c33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620106/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620106/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23577158$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shih, Kuo-Shun</creatorcontrib><creatorcontrib>Lin, Chih-Chang</creatorcontrib><creatorcontrib>Hung, Hsiao-Fang</creatorcontrib><creatorcontrib>Yang, Yu-Chi</creatorcontrib><creatorcontrib>Wang, Chung-An</creatorcontrib><creatorcontrib>Jeng, Kee-Ching</creatorcontrib><creatorcontrib>Fu, Hua-Wen</creatorcontrib><title>One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.</description><subject>Anion exchanging</subject><subject>Anion-exchange chromatography</subject><subject>Antigens</subject><subject>Aquaporins</subject><subject>Asthma</subject><subject>Bacillus subtilis</subject><subject>Bacillus subtilis - genetics</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - isolation & purification</subject><subject>Bacterial Proteins - pharmacology</subject><subject>Biology</subject><subject>Bladder</subject><subject>Bladder cancer</subject><subject>Cancer</subject><subject>Care and treatment</subject><subject>Cellular biology</subject><subject>Chemical compounds</subject><subject>Chemistry</subject><subject>Chromatography</subject><subject>Chromatography, Ion Exchange - methods</subject><subject>Columns (structural)</subject><subject>Cytokines</subject><subject>Dextrans</subject><subject>Diagnosis</subject><subject>Diagnostic reagents</subject><subject>Diagnostic systems</subject><subject>Drug development</subject><subject>Drugs</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Gastric cancer</subject><subject>Gene Expression</subject><subject>Health aspects</subject><subject>Helicobacter infections</subject><subject>Helicobacter pylori</subject><subject>Humans</subject><subject>Impurities</subject><subject>Infection</subject><subject>Infections</subject><subject>Inflammation</subject><subject>Leukocytes (neutrophilic)</subject><subject>Medicine</subject><subject>Neutrophils</subject><subject>Neutrophils - drug effects</subject><subject>Neutrophils - metabolism</subject><subject>Oxygen</subject><subject>pH effects</subject><subject>Pharmacology</subject><subject>Protein binding</subject><subject>Protein purification</subject><subject>Protein structure</subject><subject>Proteins</subject><subject>Purification</subject><subject>Purity</subject><subject>Reactive oxygen species</subject><subject>Reactive Oxygen Species - metabolism</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - pharmacology</subject><subject>Resins</subject><subject>Secondary structure</subject><subject>Stomach cancer</subject><subject>Tumor necrosis factor-TNF</subject><subject>Ulcers</subject><subject>Urinary bladder</subject><subject>Vaccines</subject><subject>Virulence</subject><subject>Virulence factors</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk01v1DAQhiMEoqXwDxBEQkJw2MUfsZ1ckEoFdKVKK_F1tRxnknXljYPtVN1_j5dNqw3qAfkQe_LMO_E7mSx7idESU4E_XLvR98ouB9fDEiGORMkfZae4omTBCaKPj_Yn2bMQrhFitOT8aXZCKBMCs_I02617WIQIQ6433m1VdJ1Xw8bofBi9aY1W0bg-d21-CdZoVysdwefDzjpv8h7G6F3C7SLFzU2C-y4fvItg-hxuBw8hQJOnwyeljbVjyMNYR2NNeJ49aZUN8GJ6nmU_v3z-cXG5uFp_XV2cXy00r0hcFKxgCFgjACPBMYhKsLJUNaeoKESJ2rZiSDPKyhYVpKBUkwpxUUNJ2ppoSs-y1wfdwbogJ9eCxJSismIEs0SsDkTj1LUcvNkqv5NOGfk34HwnlY9GW5CkxqIuFBecqII1TDUCCdJgomqC2qJNWh-namO9hUZDH72yM9H5m95sZOduJE19wogngXeTgHe_RwhRbk3QYK3qwY377yZcEFYWVULf_IM-fLuJ6lS6gOlbl-rqvag8TwbSUiTTErV8gEqrgW1qew-tSfFZwvtZQmIi3MZOjSHI1fdv_8-uf83Zt0fsBpSNm-DsuP8NwxwsDqD2LgQP7b3JGMn9hNy5IfcTIqcJSWmvjht0n3Q3EvQPJ7wMmQ</recordid><startdate>20130408</startdate><enddate>20130408</enddate><creator>Shih, Kuo-Shun</creator><creator>Lin, Chih-Chang</creator><creator>Hung, Hsiao-Fang</creator><creator>Yang, Yu-Chi</creator><creator>Wang, Chung-An</creator><creator>Jeng, Kee-Ching</creator><creator>Fu, Hua-Wen</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</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>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</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>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130408</creationdate><title>One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis</title><author>Shih, Kuo-Shun ; Lin, Chih-Chang ; Hung, Hsiao-Fang ; Yang, Yu-Chi ; Wang, Chung-An ; Jeng, Kee-Ching ; Fu, Hua-Wen</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-45450e5d7e10761e797588ab63044780ff950c5358f042433c29067be82fb2c33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Anion exchanging</topic><topic>Anion-exchange chromatography</topic><topic>Antigens</topic><topic>Aquaporins</topic><topic>Asthma</topic><topic>Bacillus subtilis</topic><topic>Bacillus subtilis - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shih, Kuo-Shun</au><au>Lin, Chih-Chang</au><au>Hung, Hsiao-Fang</au><au>Yang, Yu-Chi</au><au>Wang, Chung-An</au><au>Jeng, Kee-Ching</au><au>Fu, Hua-Wen</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2013-04-08</date><risdate>2013</risdate><volume>8</volume><issue>4</issue><spage>e60786</spage><epage>e60786</epage><pages>e60786-e60786</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23577158</pmid><doi>10.1371/journal.pone.0060786</doi><tpages>e60786</tpages><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_1330895215 |
| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Anion exchanging Anion-exchange chromatography Antigens Aquaporins Asthma Bacillus subtilis Bacillus subtilis - genetics Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - isolation & purification Bacterial Proteins - pharmacology Biology Bladder Bladder cancer Cancer Care and treatment Cellular biology Chemical compounds Chemistry Chromatography Chromatography, Ion Exchange - methods Columns (structural) Cytokines Dextrans Diagnosis Diagnostic reagents Diagnostic systems Drug development Drugs E coli Escherichia coli Gastric cancer Gene Expression Health aspects Helicobacter infections Helicobacter pylori Humans Impurities Infection Infections Inflammation Leukocytes (neutrophilic) Medicine Neutrophils Neutrophils - drug effects Neutrophils - metabolism Oxygen pH effects Pharmacology Protein binding Protein purification Protein structure Proteins Purification Purity Reactive oxygen species Reactive Oxygen Species - metabolism Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - pharmacology Resins Secondary structure Stomach cancer Tumor necrosis factor-TNF Ulcers Urinary bladder Vaccines Virulence Virulence factors |
| title | One-step chromatographic purification of Helicobacter pylori neutrophil-activating protein expressed in Bacillus subtilis |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-05T08%3A09%3A19IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=One-step%20chromatographic%20purification%20of%20Helicobacter%20pylori%20neutrophil-activating%20protein%20expressed%20in%20Bacillus%20subtilis&rft.jtitle=PloS%20one&rft.au=Shih,%20Kuo-Shun&rft.date=2013-04-08&rft.volume=8&rft.issue=4&rft.spage=e60786&rft.epage=e60786&rft.pages=e60786-e60786&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0060786&rft_dat=%3Cgale_plos_%3EA478387433%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1330895215&rft_id=info:pmid/23577158&rft_galeid=A478387433&rft_doaj_id=oai_doaj_org_article_2b17b4a6762a45d5ad7072d12ab20f4f&rfr_iscdi=true |