Advantages and Disadvantages of His-Tagged Beta-Galactosidase

Advantages and Disadvantages of His-Tagged Beta-Galactosidase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Advantages and Disadvantages of His-Tagged Beta-Galactosidase Sandra S. Flores, Pedro D. Clop, José L. Barra, Carlos Argaraña, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-54150/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract β-Galactosidase is one of the most important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation and evidenced greater half-life time compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Analytical ultracentrifugation experiments demonstrated that the population of β-GalHis molecules exhibited a higher proportion of monomers and a lower proportion of tetrameric species with respect to the His-tag free protein. The impairment of tetramerization may would explain the negative effect of the presence of His-tag on the enzymatic activity. In addition, the present results, analyzed in the context of the available literature, suggest that the effect of the His-tag is protein-specific. Applied & Industrial Microbiology beta-galactosidase His-tag enzymatic activity protein structure thermal stability Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-54150","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":1398472,"identity":"15af8c3a-dbc2-49eb-b06a-97da443c0257","order_by":0,"name":"Sandra S. Flores","email":"","orcid":"","institution":"Universidad Nacional de Cordoba Facultad de Ciencias Exactas Fisicas y Naturales. ICTA. Depto de Química. CONICET. 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Sanchez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBACxgbmhgMwDjMQy0GYbPi0MAK1JCC0GBPUAtLEgKwlsYGQFuYZiY0HPv6wY5BvP3vwc0FFXfqG280PGD6UHWbgn30AqxbGGYkNB2ckJDMYnMlLlp5xhi13w51jBowzzh1mkDiXgFPLYZ4EZgYDhhwzZt42ntwNN3IYgIzDDAxncPgEpOVPQj2DfP8boJZ/EukGIC1/gVrk8WlhSACaeQNkS4NBAlgLI1CLAS4tPQ8bDvakHecxuPHGWJrnWILhzBtpBgd7zqXzGOLQYtiefPjDD5tqOfn+HMPPPDV18nw3kh8++FFmLSeHS0sDhOZBET2AIYIE5HFJjIJRMApGwSiAAwAD5F67QMrHQgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6676-5776","institution":"Consejo Nacional de Investigaciones Cientificas y Tecnicas y Universidad Nacional de Córdoba","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Julieta","middleName":"M.","lastName":"Sanchez","suffix":""}],"badges":[],"createdAt":"2020-08-05 11:19:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-54150/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-54150/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1955805,"identity":"26dc7bb3-77f9-48d7-a4c5-3843becc3cac","added_by":"auto","created_at":"2020-08-17 16:30:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19010,"visible":true,"origin":"","legend":"Functional β-Gal properties. a) Specific activity as a function of lactose concentration (β-GalWTwhite symbols, β-GalHis, black symbols). Samples were incubated for 20 min, at 37°C and pH 6.8 and all measurements were carried out in conditions of initial velocity, according to Michaelis-Menten (MM) model. Hyperbolic curves could be adjusted to the experimental points, according to the MM equation. The resulting kinetic parameters are shown in Table 1.b) β-Gals SDS-PAGE. ","description":"","filename":"Fig.JPG","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1/Fig.JPG"},{"id":1955806,"identity":"161d4a10-f5ba-40a1-9c38-a13d5ec9c2cd","added_by":"auto","created_at":"2020-08-17 16:30:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31672,"visible":true,"origin":"","legend":" Thermal and temporal inactivation profiles of -Gals.a) Specific activity measured at the indicated temperature within the range 22ºC - 65ºC. b) β-Gal specific activity measured at 37ºC after preincubation for 20 min. at different temperatures (20-60ºC). In each data set normalization was done with respect to the corresponding specific activity preincubated at 30ºC, which was taken as the unity. c) Catalytic activity was evaluated at 37ºC, pH 6.8 and lactose 200 mM after preheating the proteins (0.01 g/L) at 50ºC during a fix time period (20-60 min). Exponential decay curves (dashed lines) are the fitness to the experimental points (black and white circles) and allowed estimating the half-life times (shown in Table 1). ","description":"","filename":"Fig2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1/Fig2.JPG"},{"id":1955807,"identity":"3352309b-1b2b-4bc7-ba3b-abe96da9b433","added_by":"auto","created_at":"2020-08-17 16:30:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18783,"visible":true,"origin":"","legend":"Structural analysis of -Gals a) Intrinsic fluorescence spectra of of -GalHis (dotted line) and -GalWT (full line) proteins at 0.2 g/L at 25°C. b) Effect of temperature on the max of -GalHis (⚫) and -GalWT (). ","description":"","filename":"Fig3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1/Fig3.JPG"},{"id":1955808,"identity":"e5847896-b70f-47d2-8024-0387b8b6b9a0","added_by":"auto","created_at":"2020-08-17 16:30:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13731,"visible":true,"origin":"","legend":"Analytic ultracentrifugation of β-GalHis (full line) and β-GalWT (dotted line) ","description":"","filename":"Fig4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1/Fig4.JPG"},{"id":13523509,"identity":"07a2ab77-b151-481d-a2f0-254fe46eedfa","added_by":"auto","created_at":"2021-09-17 00:40:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":564032,"visible":true,"origin":"","legend":"","description":"","filename":"FloresSS2020agostofinalfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1_covered.pdf"},{"id":1955820,"identity":"b3840ab0-903c-48d4-8428-097c811213bc","added_by":"auto","created_at":"2020-08-17 16:30:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":657290,"visible":true,"origin":"","legend":"","description":"","filename":"FloresSS2020agostofinalfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1_stamped.pdf"},{"id":1955809,"identity":"97562c92-23b6-4226-a438-1fee6478dfb9","added_by":"auto","created_at":"2020-08-17 16:30:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":592682,"visible":true,"origin":"","legend":"","description":"","filename":"FloresSS2020agostofinalfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-54150/v1/FloresSS2020agostofinalfinal.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eAdvantages and Disadvantages of His-Tagged Beta-Galactosidase\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-54150/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"beta-galactosidase, His-tag, enzymatic activity, protein structure, thermal stability","lastPublishedDoi":"10.21203/rs.3.rs-54150/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-54150/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"β-Galactosidase is one of the most important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant β-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (β-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (β-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of β-GalHis with respect to β-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, β-GalHis presented a higher resistance to the thermal inactivation and evidenced greater half-life time compared to β-GalWT. At room temperature, β-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein however, it exhibited a lower tendency to the thermal-induced unfolding with respect to β-GalWT. Analytical ultracentrifugation experiments demonstrated that the population of β-GalHis molecules exhibited a higher proportion of monomers and a lower proportion of tetrameric species with respect to the His-tag free protein. The impairment of tetramerization may would explain the negative effect of the presence of His-tag on the enzymatic activity. In addition, the present results, analyzed in the context of the available literature, suggest that the effect of the His-tag is protein-specific.","manuscriptTitle":"Advantages and Disadvantages of His-Tagged Beta-Galactosidase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-08-17 16:14:34","doi":"10.21203/rs.3.rs-54150/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba7635ca-3925-4b6b-956c-44045925bf61","owner":[],"postedDate":"August 17th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":306259,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2020-09-04T20:59:09+00:00","versionOfRecord":[],"versionCreatedAt":"2020-08-17 16:14:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-54150","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-54150","identity":"rs-54150","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}