Histidine-Tagged Recombinant Protein Purification and On-Column Refolding

Print

Overview

Bacteria are widely used as hosts for the production of recombinant proteins that do not require posttranslational modifications such as glycosylation for bioactivity. In this section, we describe the purification of a Staphylococcus aureus α-hemolysin protein from E. coli. This protein is a 293-amino-acid toxin that is not easily purified. Here, we describe a single-step protocol used to purify and refold α-hemolysin that is produced as inclusion bodies in E. coli. Other methods of protein purification rely on multiple steps for the recovery of bioactive proteins from inclusion bodies, where the most important step is the refolding of the protein into a bioactive form. Protein expression in the form of inclusion bodies is often considered undesirable because the protein is insoluble. To solubilize proteins from inclusion bodies, the proteins must be dissolved and unfolded, thereby incurring the risk of destroying the activity of the protein. In this application, we describe how denaturation is minimized through controlled on-column refolding.

 

Related Topics: Antibody Purification, Disease Diagnosis Using Chromatography, Chromatography in Food Production, Analysis of Wine Fermentation Using Chromatography, and Protein Purification and Isolation.

 
 
Page Contents
 
 
 

Protein Expression

In contrast to the risk of denaturation during solubilization, protein expression as inclusion bodies presents several advantages:

  • Expression at very high levels, representing up to 30% of cellular protein
  • Easy isolation of the inclusion bodies due to differences in size and density compared with other contaminants
  • Low degradation of the expressed protein due to the inclusion body’s resistance to proteolysis
  • High homogeneity of the protein of interest in the cell lysate, which helps to reduce the number of purification steps
  • The ability to overexpress toxic proteins, especially in large-scale production

After pelleting cells by centrifugation, the membrane proteins are solubilized, and the inclusion bodies are recovered by further centrifugation and solubilization. The final soluble fraction is clarified by a third centrifugation spin before loading onto a Bio-Scale™ Mini Profinity™ IMAC column, where refolding occurs during an integrated washing step. The protein of interest is then eluted and subsequently desalted by passage through a second column, a Bio-Scale™ Mini Bio-Gel® P-6 column (figure 1).

SDS-PAGE gel showing the production and purification of refolded His-αHA using the native IMAC + desalting method on the Profinia system.

Fig. 1. SDS-PAGE gel showing the production and purification of refolded His-αHA using the native IMAC + desalting method on the Profinia™ system. Lane M, molecular weight markers; lane 1, E. coli total extracts containing overexpressed insoluble His-αHA; lane 2, extracted and urea-solubilized inclusion bodies; lane 3, purified and refolded His-αHA. Only one-third of the sample that is equivalent to lane 2 was loaded. (Rhazi et al. 2009).

This method allows the user to rapidly obtain purified bioactive staphylococcal α-hemolysin toxin in a single step, saving significant time over other methods, which can require up to 60 hours. Because the protein contains a polyhistidine tag, it is amenable to on-column refolding and purification in a single step. The unfolded protein binds to the column due to the presence of denaturant in the binding buffer. Subsequent washing and elution steps use buffers that do not contain urea, the denaturant, which allows the protein to refold on the column. Refolding proteins on the column is efficient because it minimizes intermolecular interactions that can lead to protein aggregation. The proper folding of the protein is confirmed using a functional assay, wherein the
α-hemolysin is tested for hemolytic activity against erythrocytes (figure 2). Using this method, 3.5 mg of histidine-tagged α-hemolysin can be purified and refolded in less than one hour and the purified protein is free of precipitate, indicating that no aggregation occurs.

Hemolytic activity of His-aHA

Fig. 2. Hemolytic activity of His-αHA. 1, hemolytic assay performed with the His-aHA prepared using a drop-wise dilution method. 2, hemolytic assay with refolded His-αHA purified with the Profinia protein purification system. The assays were performed by incubation of the toxin (20 µg/ml) with 1 ml of a 5% defibrinated rRBC suspension in PBS, pH 7.3, for 30 min at 37°C. The hemoglobin release was calculated from absorbance measurements at 540 nm. (Rhazi et al. 2009).

 
MA7DZ94VY [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [54.36.148.79, 10.232.18.243] [accept] = [*/*] [seourl] = [/en-us/applications-technologies/affinity-chromatography] [x-amzn-trace-id] = [Root=1-5c13a012-e688e8e212fb57f67ad97e72] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=MA7DZ94VY] [host] = [10.232.17.28:1776] [x-request-uri] = [/en-us/applications-technologies/affinity-chromatography] [connection] = [Keep-Alive] [accept-encoding] = [deflate, gzip] [user-agent] = [Mozilla/5.0 (compatible; AhrefsBot/5.2; +http://ahrefs.com/robot/)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/affinity-chromatography LSR MA7DZ94VY Polyhistidine-Tagged Recombinant Protein Purification and On-Column Refolding Histidine-Tagged Recombinant Protein Purification and On-Column Refolding /webroot/web/html/lsr/solutions/applications/protein_purification <p>Bacteria are widely used as hosts for the production of recombinant proteins that do not require posttranslational modifications such as glycosylation for bioactivity. In this section, we describe the purification of a <em>Staphylococcus aureus</em> &alpha;-hemolysin protein from <em>E. coli</em>. This protein is a 293-amino-acid toxin that is not easily purified. Here, we describe a single-step protocol used to purify and refold &alpha;-hemolysin that is produced as inclusion bodies in <em>E. coli</em>. Other methods of protein purification rely on multiple steps for the recovery of bioactive proteins from inclusion bodies, where the most important step is the refolding of the protein into a bioactive form. Protein expression in the form of inclusion bodies is often considered undesirable because the protein is insoluble. To solubilize proteins from inclusion bodies, the proteins must be dissolved and unfolded, thereby incurring the risk of destroying the activity of the protein. In this application, we describe how denaturation is minimized through controlled on-column refolding.</p> <p>&nbsp;</p> <p><strong>Related Topics: </strong><a href="/evportal/destination/solutions?catID=MA7DUU15">Antibody Purification</a>, <a href="/evportal/destination/solutions?catID=MA7JNPE8Z">Disease Diagnosis Using Chromatography</a>, <a href="/evportal/destination/solutions?catID=MA7JLH15">Chromatography in Food Production</a>, <a href="/evportal/destination/solutions?catID=MA7JPD4VY">Analysis of Wine Fermentation Using Chromatography</a>, and <a href="/en-us/applications-technologies/protein-purification-isolation"> Protein Purification and Isolation</a>.</p> Protein Expression <p>In contrast to the risk of denaturation during solubilization, protein expression as inclusion bodies presents several advantages:</p> <ul> <li>Expression at very high levels, representing up to 30% of cellular protein </li> <li>Easy isolation of the inclusion bodies due to differences in size and density compared with other contaminants</li> <li>Low degradation of the expressed protein due to the inclusion body&rsquo;s resistance to proteolysis</li> <li>High homogeneity of the protein of interest in the cell lysate, which helps to reduce the number of purification steps</li> <li>The ability to overexpress toxic proteins, especially in large-scale production</li> </ul> <p>After pelleting cells by centrifugation, the membrane proteins are solubilized, and the inclusion bodies are recovered by further centrifugation and solubilization. The final soluble fraction is clarified by a third centrifugation spin before loading onto a <a href="/evportal/destination/commerce/product_detail?catID=83fa1b11-6e0e-468d-b4e0-7c2bbe5bbd9b">Bio-Scale&trade; Mini Profinity&trade; IMAC column</a>, where refolding occurs during an integrated washing step. The protein of interest is then eluted and subsequently desalted by passage through a second column, a <a href="/evportal/destination/commerce/product_detail?catID=2cca3bca-ca44-4935-9314-0119b1be4b9b">Bio-Scale&trade; Mini Bio-Gel<sup>&reg;</sup> P-6 column</a> (figure 1).</p> <p><img src="/webroot/web/images/lsr/solutions/applications/protein_purification_and_isolation/application_detail/sds-page-gel-showing-production-purification-of-refolded-his-aha.jpg" alt="SDS-PAGE gel showing the production and purification of refolded His-&alpha;HA using the native IMAC + desalting method on the Profinia system." width="363" height="283" /></p> <p class="caption"><strong>Fig. 1. SDS-PAGE gel showing the production and purification of refolded His-&alpha;HA using the native IMAC + desalting method on the Profinia&trade; system</strong>. Lane M, molecular weight markers; lane 1, <em>E. coli</em> total extracts containing overexpressed insoluble His-&alpha;HA; lane 2, extracted and urea-solubilized inclusion bodies; lane 3, purified and refolded His-&alpha;HA. Only one-third of the sample that is equivalent to lane 2 was loaded. (<a style="font-size: 10px" href="#references">Rhazi et al. 2009</a>).</p> <p>This method allows the user to rapidly obtain purified bioactive staphylococcal &alpha;-hemolysin toxin in a single step, saving significant time over other methods, which can require up to 60 hours. Because the protein contains a polyhistidine tag, it is amenable to on-column refolding and purification in a single step. The unfolded protein binds to the column due to the presence of denaturant in the binding buffer. Subsequent washing and elution steps use buffers that do not contain urea, the denaturant, which allows the protein to refold on the column. Refolding proteins on the column is efficient because it minimizes intermolecular interactions that can lead to protein aggregation. The proper folding of the protein is confirmed using a functional assay, wherein the<br /> &alpha;-hemolysin is tested for hemolytic activity against erythrocytes (figure 2). Using this method, 3.5 mg of histidine-tagged &alpha;-hemolysin can be purified and refolded in less than one hour and the purified protein is free of precipitate, indicating that no aggregation occurs.</p> <p><img src="/webroot/web/images/lsr/solutions/applications/protein_purification_and_isolation/application_detail/hemolytic-activity-of-his-aha.jpg" alt="Hemolytic activity of His-aHA" width="489" height="212" /></p> <p class="caption"><strong>Fig. 2. Hemolytic activity of His-&alpha;HA.</strong> 1, hemolytic assay performed with the His-aHA prepared using a drop-wise dilution method. 2, hemolytic assay with refolded His-&alpha;HA purified with the Profinia protein purification system. The assays were performed by incubation of the toxin (20 &micro;g/ml) with 1 ml of a 5% defibrinated rRBC suspension in PBS, pH 7.3, for 30 min at 37&deg;C. The hemoglobin release was calculated from absorbance measurements at 540 nm. (<a style="font-size: 10px" href="#references">Rhazi et al. 2009</a>).</p> References <p><a name="references"></a></p> <p>Rhazi N et al. (2009). <a href="/webroot/web/pdf/lsr/literature/Bulletin_5870.pdf" target="_blank">Purification and on-column refolding of polyhistidine-tagged <em>Staphylococcus aureus</em> &alpha;-hemolysin inclusion bodies on the Profinia&trade; protein purification system</a>. Bio-Rad Bulletin 5870.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Life Science Research/Products/Chromatography/Affinity Chromatography and Tag Cleavage Kit Products/General Affinity Kits ->MTS::KXNH0H15##Life Science Research/Products/Chromatography/Chromatography Media/Cation Exchange Chromatography Media/UNOsphere Cation Exchange Media ->MT::475eb93b-673a-40f8-a055-7a05d41be4b3##Life Science Research/Products/Chromatography/Chromatography Media/Affinity Chromatography Media/Profinity IMAC Resins ->MT::e54ab03c-d281-4e6b-aa0d-193b7737626d##Life Science Research/Products/Chromatography/Chromatography Media/Anion Exchange Chromatography Media/UNOsphere Anion Exchange Media ->MT::36286010-253e-4553-b13d-8b5917f8e93a## Karen Moss Histidine-Tagged Recombinant Protein Purification 09/19/12 10:53 AM 09/19/22 01:13 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Applications/Protein_Purification_and_Isolation N 0
Sign Up for Bio-Rad Updates!
Enter your email address below to receive your choice of the latest news, promotions, and more.
MA7DZ94VY [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [3.80.85.76, 10.232.3.53] [accept] = [text/html,application/xhtml+xml,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/histidine-tagged-recombinant-protein-purification-column-refolding] [x-amzn-trace-id] = [Root=1-5c14323a-b4926c60b90bfda0a54171c0] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=MA7DZ94VY] [host] = [10.232.17.28:1776] [x-request-uri] = [/en-us/applications-technologies/histidine-tagged-recombinant-protein-purification-column-refolding] [connection] = [Keep-Alive] [accept-encoding] = [gzip] [user-agent] = [CCBot/2.0 (https://commoncrawl.org/faq/)] AppTech/AppTechDetails pageStyleKey applications-technologies/histidine-tagged-recombinant-protein-purification-column-refolding LSR 03/26/14 02:17 PM en_US true internet/generic 2018-10-10 22:22:20 /default/main/bio-rad/STAGING 1385504243813 US templatedata/internet/generic/data/en_US/GNL/page_generic_footer_b2c_1385505294_US.xml page_generic_footer_b2c /default/main/bio-rad/WORKAREA/default jc7v8okm 2017-10-27 13:13:40 true Done jc7v8okm templatedata/internet/generic/data/en_US/GNL/page_generic_footer_b2c_1385505294_US.xml GNL N /default/main/bio-rad en_US 11/27/23 02:18 PM 1532127468176 2018-10-10 22:22:20 /default/main/bio-rad/WORKAREA/default/templatedata/internet/generic/data/en_US/GNL/page_generic_footer_b2c_1385505294_US.xml footer-page 1 1385504243813 page_generic_footer_b2c <div class="links"> <h4>Support</h4> <ul> <li><a class="msdsLink wtclass linkgeneration" href="/_locale/literature-library">MSDS</a></li> <li><a class="cofaLink wtclass linkgeneration" href="/_locale/_defaultVerticalUrl/support/certificate-of-analysis">Certificate of Analysis</a></li> <li><a class="qcinsertsLink wtclass" href="http://myeinserts.qcnet.com/" target="_blank" rel="noopener noreferrer">QC Inserts</a></li> <li><a class="technicalsupportLink wtclass linkgeneration" href="/_locale/life-science-research/support">Technical Support<br />For Life Science</a></li> <li><a class="expertCSLink wtclass linkgeneration" href="/_locale/_defaultVerticalUrl/purchase-service-programs/expert-care-service?ID=1383780912356">Expert Care Service</a></li> <li><a class="feedBackLink wtclass linkgeneration" id="feedbackID" href="javascript:void(0)">Feedback</a></li> <li><a class="contactUSLink wtclass linkgeneration" href="/_locale/contact-us">Contact Us</a></li> </ul> </div> <div class="links"> <h4>Ordering</h4> <ul> <li><a id="quickOrderLink" class="quickOrderLink wtclass" name="quickOrderLink" href="javascript:openAjaxOverlay('/evportal/portlets/ordering/quickOrderOverlayContent.jsp?quickorder_location=Footer')">Quick Order</a></li> <li><a class="mybioradLink wtclass linkgeneration" href="/_locale/my-bio-rad">My Bio-Rad</a></li> <li><a id="footer_orderLink" class="orderhistoryLink wtclass" href="javascript:void(0);">Order History</a></li> <li><a id="footer_quoteLink" href="javascript:void(0);">Quote History</a></li> <li><a class="supplycentersLink wtclass linkgeneration" href="/_locale/_defaultVerticalUrl/purchase-service-programs/supply-center-program">Supply Centers</a></li> <li><a class="returnpolicyLink wtclass linkgeneration" href="javascript:openAjaxOverlay('/webroot/web/html/returnPolicy-us.html','Return and Refund Policy');">Return and Refund Policy</a></li> </ul> </div> <div class="links"> <h4>Our Products</h4> <ul> <li><a class="lsrLink wtclass linkgeneration" href="/_locale/life-science-research">Life Science Research</a></li> <li><a class="cdLink wtclass linkgeneration" href="/_locale/clinical-diagnostics">Clinical Diagnostics</a></li> <li><a class="infLink wtclass linkgeneration" href="/_locale/spectroscopy">Spectroscopy</a></li> <li><a class="psLink wtclass linkgeneration" href="/_locale/process-separations">Process Separations</a></li> <li><a class="fsLink wtclass linkgeneration" href="/_locale/food-science">Food Science</a></li> <li><a class="lseLink wtclass linkgeneration" href="/_locale/education">Life Science Education</a></li> <li><a class="selectionguideLink wtclass linkgeneration" href="/featured/en/selection-guides.html">Selection Guides</a></li> <li><a class="featuredproductsLink wtclass" href="/featured/en/featured-products.html">Featured Products</a></li> </ul> </div> <div class="links links2"> <h4>Corporate</h4> <ul> <li><a class="aboutbioradLink wtclass linkgeneration" href="/_locale/corporate/about-bio-rad">About Bio-Rad</a></li> <li><a class="careersLink wtclass linkgeneration" href="/_locale/corporate/careers">Careers</a></li> <li><a class="irLink wtclass linkgeneration" href="/_locale/corporate/investor-relations">Investor Relations</a></li> <li><a class="coLink wtclass linkgeneration" href="/_locale/corporate/community-outreach">Community Outreach</a></li> <li><a class="pteLink wtclass linkgeneration" href="/_locale/corporate/protecting-environment">Protecting the Environment</a></li> <li><a class="newsroomLink wtclass linkgeneration" href="/_locale/corporate/newsroom">Newsroom</a></li> </ul> </div> <p> <script type="text/javascript">// <![CDATA[ $(document).ready(function(){ setSterlingUrlsToHtmlHrefVariables(); $('a.linkgeneration').each(function(){ $(this).attr('href', $(this).attr('href').replace('_locale', languageCode + '-' + countryCode.toLowerCase()).replace('_verticalUrl', currentVerticalUrlTitle).replace('_defaultVerticalUrl', defaultVerticalUrlTitle).replace('_feedbackCMSID',feedbackCMSID)); }); var feedbackOverlayUrl = '/bio-rad/Standalone/FeedbackOverlay.page?standalonePage=true'; $("#feedbackID").click(function(){ openAjaxOverlay(feedbackOverlayUrl,"Feedback"); }); }); // ]]></script> <script type="text/javascript">// <![CDATA[ if($.cookie('evCntryLang')){ (function(){"use strict";var e=null,b="4.0.0", n="10740", additional="", t,r,i;try{t=top.document.referer!==""?encodeURIComponent(top.document.referrer.substring(0,2048)):""}catch(o){t=document.referrer!==null?document.referrer.toString().substring(0,2048):""}try{r=window&&window.top&&document.location&&window.top.location===document.location?document.location:window&&window.top&&window.top.location&&""!==window.top.location?window.top.location:document.location}catch(u){r=document.location}try{i=parent.location.href!==""?encodeURIComponent(parent.location.href.toString().substring(0,2048)):""}catch(a){try{i=r!==null?encodeURIComponent(r.toString().substring(0,2048)):""}catch(f){i=""}}var l,c=document.createElement("script"),h=null,p=document.getElementsByTagName("script"),d=Number(p.length)-1,v=document.getElementsByTagName("script")[d];if(typeof l==="undefined"){l=Math.floor(Math.random()*1e17)}h="dx.steelhousemedia.com/spx?"+"dxver="+b+"&shaid="+n+"&tdr="+t+"&plh="+i+"&cb="+l+additional;c.type="text/javascript";c.src=("https:"===document.location.protocol?"https://":"http://")+h;v.parentNode.insertBefore(c,v)})() } // ]]></script> </p> /webroot/web/html footer_page_b2c_en_US.html /webroot/web/html/footer_page_b2c_en_US.html Karen Moss Footer page Footer 03/26/14 02:17 PM 11/27/23 02:18 PM US en GNL /GNL N 0 en-us Home <script type="text/javascript">$(document).ready(function() { $(".footer-nav > a:nth-child(3)").after(" | <a href='javascript:void(0)' id='regionlink'>Web Accessibility</a>"); var region = $("#headlangID .lang strong").html(); $("#regionlink").click(function(){ window.location.replace( "/webroot/web/html/featured/en/web-accessibility.html?Region=" + region );});});</script>Trademarks Site Terms Terms & Conditions Imprint EU Recycle Program Privacy Change Cookie Setting <script type="text/javascript">$(document).ready(function () {$(".links2").append('<style>.social-links-br {float: left;margin-right: 10px;}.social-links-br img {width: 20px;height: 20px;margin-right: 5px}</style><div class="social-links-br"><a class="no-target-style" href="https://www.linkedin.com/company/bio-rad?trk=top_nav_home" target="_blank"><img src="/webroot/web/images/general/icons/linkedin_round_40x40.png" alt="Bio-Rad LinkedIn"></a><a class="no-target-style" href="https://www.youtube.com/user/BioRadLifeScience" target="_blank"><img src="/webroot/web/images/general/icons/youtube-icon-rnd-40x40.png" alt="Bio-Rad YouTube"></a><a class="no-target-style" href="https://twitter.com/BioRadLifeSci" target="_blank"><img src="/webroot/web/images/general/icons/twitter_round_40x40.png" alt="Bio-Rad Twitter"></a><a class="no-target-style" href="https://www.facebook.com/biorad/" target="_blank"><img src="/webroot/web/images/general/icons/facebook-icon-rnd-40x40.png" alt="Bio-Rad Facebook"></a><a class="no-target-style" href="https://www.instagram.com/bioradlabs/" target="_blank"><img src="/webroot/web/images/general/icons/instagram-icon-rnd-40x40.png" alt="Bio-Rad Instagram"></a></div>');});</script>Copyright &copy; {0} Bio-Rad Laboratories, Inc. All rights reserved. This page was last edited on {0} True GTM-MJ2DN2S us US en LSR life-science-research life-science-research /prd/en/US/direct/biorad?ts=1&cmd=OrdersWorkspaceDisplay /prd/en/US/direct/biorad?ts=1&cmd=QuotesWorkspaceDisplay /prd/en/US/direct/biorad?ts=1&cmd=InvoiceWorkspaceDisplay&_Tab=Invoice false false true false true true 1040988879 LHsYCOu_wQMQz_Ww8AM window.google_tag_params true 272-THL-329 username CartID_ WT_FPC <script type="text/javascript">$.ajax({url: document.location.protocol + '//munchkin.marketo.net/munchkin.js',dataType: 'script',cache: true,success: function() {Munchkin.init('044-CBN-599');}});</script> <script type="text/javascript" src="/evportal/framework/skins/evolution/js/proxyMunchkin.js"></script> 2018