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Seraphine V. Wegner - One of the best experts on this subject based on the ideXlab platform.
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Multifunctional streptavidin–biotin conjugates with precise stoichiometries
Chemical Science, 2020Co-Authors: Seraphine V. WegnerAbstract:Streptavidin is ubiquitously used to link different biotinylated molecules thanks to its tetravalent binding to biotin. An unwanted side-effect is the resulting statistical mixtures of products. Here, a general approach to form multifunctional streptavidin conjugates with precise stoichiometries and number of open binding pockets is reported. This method relies on an iminobiotin-Polyhistidine Tag, which allows separating streptavidin conjugates with different numbers of Tags, and later reopening binding pockets at lowered pH to introduce a second functionality. Pure fluorescently labelled mono-, di- and trivalent streptavidin–biotin conjugates prepared in this way were used for imaging biotinylated cell surface molecules with controlled clustering. Furthermore, these conjugates were functionalized with a second biotinylated molecule, folic acid–biotin, to investigate the importance of multivalent binding in targeted delivery of cancer cells. These streptavidin–biotin conjugates with precise stoichiometries combined with a variety of biotinylated molecules render this method a diverse and powerful tool for molecular biology and biotechnology.
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multifunctional streptavidin biotin conjugates with precise stoichiometries
Chemical Science, 2020Co-Authors: Seraphine V. WegnerAbstract:Streptavidin is ubiquitously used to link different biotinylated molecules thanks to its tetravalent binding to biotin. An unwanted side-effect is the resulting statistical mixtures of products. Here, a general approach to form multifunctional streptavidin conjugates with precise stoichiometries and number of open binding pockets is reported. This method relies on an iminobiotin-Polyhistidine Tag, which allows separating streptavidin conjugates with different numbers of Tags, and later reopening binding pockets at lowered pH to introduce a second functionality. Pure fluorescently labelled mono-, di- and trivalent streptavidin–biotin conjugates prepared in this way were used for imaging biotinylated cell surface molecules with controlled clustering. Furthermore, these conjugates were functionalized with a second biotinylated molecule, folic acid–biotin, to investigate the importance of multivalent binding in targeted delivery of cancer cells. These streptavidin–biotin conjugates with precise stoichiometries combined with a variety of biotinylated molecules render this method a diverse and powerful tool for molecular biology and biotechnology.
Nur Basak Surmeli - One of the best experts on this subject based on the ideXlab platform.
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Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119
Bioinorganic chemistry and applications, 2019Co-Authors: Yaprak Aslantas, Nur Basak SurmeliAbstract:Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine Tags (His-Tags) are widely used to simplify purification of proteins. However, His-Tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-Tags on CYP119. To this end, the His-Tags were cloned at the N-terminus or C-terminus of the CYP119, and His-Tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-Tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-Tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-Tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-Tag affects electronic properties of the active site heme iron.
Donald L. Mykles - One of the best experts on this subject based on the ideXlab platform.
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Expression of recombinant eyestalk crustacean hyperglycemic hormone from the tropical land crab, Gecarcinus lateralis, that inhibits Y-organ ecdysteroidogenesis in vitro
Molecular Biology Reports, 2008Co-Authors: Tyler P. Zarubin, Ernest S. Chang, Donald L. MyklesAbstract:Crustacean hyperglycemic hormone (CHH) is a pleiotropic neuropeptide that regulates carbohydrate and lipid metabolism, molting, reproduction, and osmoregulation in decapod crustaceans. CHH elevates glucose levels in the hemolymph by stimulating glycogenolysis in target tissues. It also inhibits ecdysteroidogenesis in the molting gland, or Y-organ (YO), possibly as a response to environmental stress. CHH acts via binding to a membrane receptor guanylyl cyclase, which is expressed in most tissues, including the YO. Large amounts of biologically active neuropeptide are required to investigate the mechanism of CHH signaling in the YO. Consequently, the eyestalk ganglia CHH (EG-CHH) isoform was cloned into a yeast ( Pichia pastoris ) expression vector to express recombinant mature peptide (rEG-CHH) with or without a C-terminal c-Myc/Polyhistidine Tag. Yeast cultures with unTagged or Tagged rEG-CHH inhibited ecdysteroidogenesis in YOs from European green crab ( Carcinus maenas ) 36% ( P
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Expression of recombinant eyestalk crustacean hyperglycemic hormone from the tropical land crab, Gecarcinus lateralis, that inhibits Y-organ ecdysteroidogenesis in vitro.
Molecular biology reports, 2008Co-Authors: Tyler P. Zarubin, Ernest S. Chang, Donald L. MyklesAbstract:Crustacean hyperglycemic hormone (CHH) is a pleiotropic neuropeptide that regulates carbohydrate and lipid metabolism, molting, reproduction, and osmoregulation in decapod crustaceans. CHH elevates glucose levels in the hemolymph by stimulating glycogenolysis in target tissues. It also inhibits ecdysteroidogenesis in the molting gland, or Y-organ (YO), possibly as a response to environmental stress. CHH acts via binding to a membrane receptor guanylyl cyclase, which is expressed in most tissues, including the YO. Large amounts of biologically active neuropeptide are required to investigate the mechanism of CHH signaling in the YO. Consequently, the eyestalk ganglia CHH (EG-CHH) isoform was cloned into a yeast (Pichia pastoris) expression vector to express recombinant mature peptide (rEG-CHH) with or without a C-terminal c-Myc/Polyhistidine Tag. Yeast cultures with unTagged or Tagged rEG-CHH inhibited ecdysteroidogenesis in YOs from European green crab (Carcinus maenas) 36% (P < 0.002) and 51% (P < 0.006), respectively. Purified Tagged EG-CHH inhibited YO ecdysteroidogenesis 32% (P < 0.002), but lacked hyperglycemic activity in vivo. This is the first report of recombinant EG-CHH inhibiting YO ecdysteroidogenesis. The data suggest that the Tagged recombinant peptide can be used to elucidate the CHH signaling pathway in the crustacean molting gland.
Henryk Kozlowski - One of the best experts on this subject based on the ideXlab platform.
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Impact of histidine spacing on modified Polyhistidine Tag – Metal ion interactions
Inorganica Chimica Acta, 2018Co-Authors: Joanna Wątły, Aleksandra Hecel, Magdalena Rowińska-Żyrek, Henryk KozlowskiAbstract:Abstract Histidine rich sequences are chosen both by nature and by molecular biologists due to their high affinity towards metal ions. In this work, we examine the affinity and binding modes of Cu 2+ , Ni 2+ and Zn 2+ towards two histidine Tags, the common His 6 -Tag (Ac-HHHHHH-NH 2 ) and its modified sequence, which also contains six histidines, but separated with two alanine residues (Ac-HAAHAAHAAHAAHAAHAA-NH 2 ). The spatial separation of histidines has an important impact on its coordination properties. Cu 2+ and Ni 2+ complexes with Ac-HHHHHH-NH 2 are more stable than those with Ac-HAAHAAHAAHAAHAAHAA-NH 2 ; the contrary is observed for Zn 2+ . In a narrow range of pH, Cu 2+ -Ac-HHHHHH-NH 2 and Ni 2+ -Ac-HHHHHH-NH 2 can even compete with the albumin-like binding of the respective metals.
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impact of histidine spacing on modified Polyhistidine Tag metal ion interactions
Inorganica Chimica Acta, 2017Co-Authors: Joanna Wątly, Aleksandra Hecel, Magdalena Rowinskażyrek, Henryk KozlowskiAbstract:Abstract Histidine rich sequences are chosen both by nature and by molecular biologists due to their high affinity towards metal ions. In this work, we examine the affinity and binding modes of Cu 2+ , Ni 2+ and Zn 2+ towards two histidine Tags, the common His 6 -Tag (Ac-HHHHHH-NH 2 ) and its modified sequence, which also contains six histidines, but separated with two alanine residues (Ac-HAAHAAHAAHAAHAAHAA-NH 2 ). The spatial separation of histidines has an important impact on its coordination properties. Cu 2+ and Ni 2+ complexes with Ac-HHHHHH-NH 2 are more stable than those with Ac-HAAHAAHAAHAAHAAHAA-NH 2 ; the contrary is observed for Zn 2+ . In a narrow range of pH, Cu 2+ -Ac-HHHHHH-NH 2 and Ni 2+ -Ac-HHHHHH-NH 2 can even compete with the albumin-like binding of the respective metals.
Yaprak Aslantas - One of the best experts on this subject based on the ideXlab platform.
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Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119
Bioinorganic chemistry and applications, 2019Co-Authors: Yaprak Aslantas, Nur Basak SurmeliAbstract:Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of “unactivated” carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine Tags (His-Tags) are widely used to simplify purification of proteins. However, His-Tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-Tags on CYP119. To this end, the His-Tags were cloned at the N-terminus or C-terminus of the CYP119, and His-Tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-Tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-Tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-Tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-Tag affects electronic properties of the active site heme iron.