Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and...
Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Carina Dey [verfasserIn] Philip Palm [verfasserIn] Lothar Elling [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2023

Schlagwörter:	galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N

Übergeordnetes Werk:	In: Molecules - MDPI AG, 2003, 28(2023), 3, p 1054
Übergeordnetes Werk:	volume:28 ; year:2023 ; number:3, p 1054

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.3390/molecules28031054

Katalog-ID:	DOAJ080615953

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520			\|a Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.
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allfields	10.3390/molecules28031054 doi (DE-627)DOAJ080615953 (DE-599)DOAJa5d7a1f659cd4c6a86abb473f71ba0e5 DE-627 ger DE-627 rakwb eng QD241-441 Carina Dey verfasserin aut Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study. galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N Organic chemistry Philip Palm verfasserin aut Lothar Elling verfasserin aut In Molecules MDPI AG, 2003 28(2023), 3, p 1054 (DE-627)311313132 (DE-600)2008644-1 14203049 nnns volume:28 year:2023 number:3, p 1054 https://doi.org/10.3390/molecules28031054 kostenfrei https://doaj.org/article/a5d7a1f659cd4c6a86abb473f71ba0e5 kostenfrei https://www.mdpi.com/1420-3049/28/3/1054 kostenfrei https://doaj.org/toc/1420-3049 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 28 2023 3, p 1054
spelling	10.3390/molecules28031054 doi (DE-627)DOAJ080615953 (DE-599)DOAJa5d7a1f659cd4c6a86abb473f71ba0e5 DE-627 ger DE-627 rakwb eng QD241-441 Carina Dey verfasserin aut Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study. galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N Organic chemistry Philip Palm verfasserin aut Lothar Elling verfasserin aut In Molecules MDPI AG, 2003 28(2023), 3, p 1054 (DE-627)311313132 (DE-600)2008644-1 14203049 nnns volume:28 year:2023 number:3, p 1054 https://doi.org/10.3390/molecules28031054 kostenfrei https://doaj.org/article/a5d7a1f659cd4c6a86abb473f71ba0e5 kostenfrei https://www.mdpi.com/1420-3049/28/3/1054 kostenfrei https://doaj.org/toc/1420-3049 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 28 2023 3, p 1054
allfields_unstemmed	10.3390/molecules28031054 doi (DE-627)DOAJ080615953 (DE-599)DOAJa5d7a1f659cd4c6a86abb473f71ba0e5 DE-627 ger DE-627 rakwb eng QD241-441 Carina Dey verfasserin aut Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study. galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N Organic chemistry Philip Palm verfasserin aut Lothar Elling verfasserin aut In Molecules MDPI AG, 2003 28(2023), 3, p 1054 (DE-627)311313132 (DE-600)2008644-1 14203049 nnns volume:28 year:2023 number:3, p 1054 https://doi.org/10.3390/molecules28031054 kostenfrei https://doaj.org/article/a5d7a1f659cd4c6a86abb473f71ba0e5 kostenfrei https://www.mdpi.com/1420-3049/28/3/1054 kostenfrei https://doaj.org/toc/1420-3049 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 28 2023 3, p 1054
allfieldsGer	10.3390/molecules28031054 doi (DE-627)DOAJ080615953 (DE-599)DOAJa5d7a1f659cd4c6a86abb473f71ba0e5 DE-627 ger DE-627 rakwb eng QD241-441 Carina Dey verfasserin aut Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study. galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N Organic chemistry Philip Palm verfasserin aut Lothar Elling verfasserin aut In Molecules MDPI AG, 2003 28(2023), 3, p 1054 (DE-627)311313132 (DE-600)2008644-1 14203049 nnns volume:28 year:2023 number:3, p 1054 https://doi.org/10.3390/molecules28031054 kostenfrei https://doaj.org/article/a5d7a1f659cd4c6a86abb473f71ba0e5 kostenfrei https://www.mdpi.com/1420-3049/28/3/1054 kostenfrei https://doaj.org/toc/1420-3049 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 28 2023 3, p 1054
allfieldsSound	10.3390/molecules28031054 doi (DE-627)DOAJ080615953 (DE-599)DOAJa5d7a1f659cd4c6a86abb473f71ba0e5 DE-627 ger DE-627 rakwb eng QD241-441 Carina Dey verfasserin aut Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays 2023 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study. galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N Organic chemistry Philip Palm verfasserin aut Lothar Elling verfasserin aut In Molecules MDPI AG, 2003 28(2023), 3, p 1054 (DE-627)311313132 (DE-600)2008644-1 14203049 nnns volume:28 year:2023 number:3, p 1054 https://doi.org/10.3390/molecules28031054 kostenfrei https://doaj.org/article/a5d7a1f659cd4c6a86abb473f71ba0e5 kostenfrei https://www.mdpi.com/1420-3049/28/3/1054 kostenfrei https://doaj.org/toc/1420-3049 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 28 2023 3, p 1054
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source	In Molecules 28(2023), 3, p 1054 volume:28 year:2023 number:3, p 1054
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topic_facet	galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N Organic chemistry
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container_title	Molecules
authorswithroles_txt_mv	Carina Dey @@aut@@ Philip Palm @@aut@@ Lothar Elling @@aut@@
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callnumber-first	Q - Science
author	Carina Dey
spellingShingle	Carina Dey misc QD241-441 misc galectin purification misc glycoprotein misc galectin fusion protein misc Gal-1 misc Gal-3 misc Gal-8N misc Organic chemistry Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays
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topic_title	QD241-441 Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays galectin purification glycoprotein galectin fusion protein Gal-1 Gal-3 Gal-8N
topic	misc QD241-441 misc galectin purification misc glycoprotein misc galectin fusion protein misc Gal-1 misc Gal-3 misc Gal-8N misc Organic chemistry
topic_unstemmed	misc QD241-441 misc galectin purification misc glycoprotein misc galectin fusion protein misc Gal-1 misc Gal-3 misc Gal-8N misc Organic chemistry
topic_browse	misc QD241-441 misc galectin purification misc glycoprotein misc galectin fusion protein misc Gal-1 misc Gal-3 misc Gal-8N misc Organic chemistry
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title	Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays
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title_full	Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays
author_sort	Carina Dey
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author_browse	Carina Dey Philip Palm Lothar Elling
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title_sort	characterization of galectin fusion proteins with glycoprotein affinity columns and binding assays
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title_auth	Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays
abstract	Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.
abstractGer	Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.
abstract_unstemmed	Galectins are β-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His<sub<6</sub<-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.
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container_issue	3, p 1054
title_short	Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays
url	https://doi.org/10.3390/molecules28031054 https://doaj.org/article/a5d7a1f659cd4c6a86abb473f71ba0e5 https://www.mdpi.com/1420-3049/28/3/1054 https://doaj.org/toc/1420-3049
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The binding characteristics to ASF of solely His<sub<6</sub<-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6–3-fold increase in binding efficiency for HSYGal-3 (His<sub<6</sub<-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His<sub<6</sub<-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His<sub<6</sub<-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. 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Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays

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