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Danny K.y. Wong - One of the best experts on this subject based on the ideXlab platform.
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Detection of estradiol at an electrochemical immunosensor with a Cu UPD|DTBP-Protein G scaffold.
Biosensors & bioelectronics, 2012Co-Authors: Xiaoqiang Liu, Heqing Feng, Xiuhua Liu, Xinhai Wang, Jiamei Zhang, Danny K.y. WongAbstract:A copper monolayer was formed on a Gold electrode surface via underpotential deposition (UPD) method to construct a Cu UPD|DTBP-Protein G immunosensor for the sensitive detection of 17β-estradiol. Copper UPD monolayer can minimize the non-specific adsorption of bioloGical molecules on the immunosensor surface and enhance the bindinG efficiency between immunosensor surface and thiolated Protein G. The crosslinker DTBP (Dimethyl 3,3'-dithiobispropionimidate · 2HCl) has stronG ability to immobilize Protein G molecules on the electrode surface and the immobilized Protein G provides an orientation-controlled bindinG of antibodies. A monolayer of propanethiol was firstly self-assembled on the Gold electrode surface, and a copper monolayer was deposited via UPD on the propanethiol modified electrode. Propanethiol monolayer helps to stabilize the copper monolayer by pushinG the formation and strippinG potentials of the copper UPD monolayer outside the potential ranGe in which copper monolayer can be damaGed easily by oxyGen in air. A droplet DTBP-Protein G was then applied on the modified electrode surface followed by the immobilization of estradiol antibody. Finally, a competitive immunoassay was conducted between estradiol-BSA (bovine serum albumin) conjuGate and free estradiol for the limited bindinG sites of estradiol antibody. Square wave voltammetry (SWV) was employed to monitor the electrochemical reduction current of ferrocenemethanol and the SWV current decreased with the increase of estradiol-BSA conjuGate concentration at the immunosensor surface. Calibration of immunosensors in waste water samples spiked with 17β-estradiol yielded a linear response up to ≈ 2200 pG mL(-1), a sensitivity of 3.20 μA/pG mL(-1) and a detection limit of 12 pG mL(-1). The favorable characteristics of the immunosensors such as hiGh selectivity, sensitivity and low detection limit can be attributed to the Cu UPD|DTBP-Protein G scaffold.
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Detection of cortisol at a Gold nanoparticle|Protein G–DTBP-scaffold modified electrochemical immunosensor
The Analyst, 2011Co-Authors: Xiaoqiang Liu, Ruoxia Zhao, Wenling Mao, Heqing Feng, Xiuhua Liu, Danny K.y. WongAbstract:An ultrasensitive electrochemical immmunosensor was demonstrated to be capable of detectinG the hormone cortisol down to concentrations as low as 16 pG mL−1. In addition, the immunosensor displayed a sensitivity of 1.6 μA pG−1 mL−1 and a linear ranGe up to ∼2500 pG mL−1 of cortisol. This immunosensor was constructed based on a Au nanoparticle|dimethyl 3,3′-dithiobispropionimidate·2HCl (DTBP)–Protein G scaffold-modified Au electrode. In this work, the Au nanoparticles were used to increase the electrochemically active surface area by 28% (with a standard deviation of 3%) to enhance the quantity of the Protein G scaffold on the electrode. Thiolation of Protein G by DTBP aided in avoidinG the confirmation chanGe of Protein G, while this Protein G–DTBP component offered an orientation-controlled immobilisation of the capture antibody on the Au electrode. In this immunosensor, a monoclonal anti-cortisol capture antibody was optimally aliGned by the scaffold before a competitive immunoassay between sample cortisol and a horseradish peroxidase-labelled cortisol conjuGate was conducted. For quantitative analysis, square wave voltammetry was used to monitor the reduction current of benzoquinone produced from a horseradish peroxidase catalysed reaction. The improved analytical performance of our immunosensor was attributed to the synerGetic effect of Au nanoparticles and the Protein G–DTBP scaffold.
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PicoGram-detection of estradiol at an electrochemical immunosensor with a Gold nanoparticle|Protein G-(LC-SPDP)-scaffold.
Talanta, 2008Co-Authors: Xiaoqiang Liu, Danny K.y. WongAbstract:Abstract Low picoGrams of the hormone 17β-estradiol were detected at an electrochemical immunosensor. This immunosensor features a Gold nanoparticle|Protein G-(LC-SPDP) 1 -scaffold, to which a monoclonal anti-estradiol capture antibody was immobilised to facilitate a competitive immunoassay between sample 17β-estradiol and a horseradish peroxidase-labelled 17β-estradiol conjuGate. Upon constructinG this molecular architecture on a disposable Gold electrode in a flow cell, amperometry was conducted to monitor the reduction current of benzoquinone produced from a catalytic reaction of horseradish peroxidase. This current was then quantitatively related to 17β-estradiol present in a sample. Calibration of immunosensors in blood serum samples spiked with 17β-estradiol yielded a linear response up to ∼1200 pG mL−1, a sensitivity of 0.61 μA/pG mL−1 and a detection limit of 6 pG mL−1. We attribute these favourable characteristics of the immunosensors to the Gold nanoparticle|Protein G-(LC-SPDP) scaffold, where the Gold nanoparticles provided a larGe electrochemically active surface area that permits immobilisation of an enhanced quantity of all components of the molecular architecture, while the Protein G-(LC-SPDP) component aided in not only reducinG steric hindrance when Protein G binds to the capture antibody, but also providinG an orientation-controlled immobilisation of the capture antibody. Coupled with amperometric detection in a flow system, the immunosensor exhibited excellent reproducibility.
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An amperometric immunosensor with a thiolated Protein G scaffold
Electrochemistry Communications, 2008Co-Authors: Jeremy Fowler, Margaret C. Stuart, Danny K.y. WongAbstract:The orientation of the analyte-specific capture antibody on the surface of electrochemical immunosensors plays an important role on their overall performance. We have employed a self-assembled layer of Protein G that was thiolated with succinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate for the orientation-controlled immobilisation of a capture antibody in a flow-type amperometric immunosensor based on a two-site sandwich immunoassay. After establishinG the formation of the thiolated Protein G layer on 1-mm screen printed electrodes, amperometric immunosensors for the detection of the hormone, human chorionic Gonadotrophin (hCG), were successfully constructed upon this scaffold. These sensors were characterised by a limit of detection (based on three times the standard deviation of the blank siGnal) of 175 IU l−1 and a linear response up to approximately 5000 IU l−1 of hCG.
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Self-assembled layer of thiolated Protein G as an immunosensor scaffold.
Analytical chemistry, 2007Co-Authors: Jeremy Fowler, Margaret C. Stuart, Danny K.y. WongAbstract:In this study, our Goal was to produce a self-assembled layer on a Gold electrode that would enable the capture of antibodies orientated for maximum bindinG to their specific antiGen in an immunosensor. To achieve this, the amine Groups from lysine residues in Protein G were initially converted to thiol Groups with 2-iminothiolane. The hiGh affinity of thiols for a Gold surface facilitates the direct formation of a self-assembled Protein G layer. FollowinG this, the coated Gold electrode was exposed to a solution of capture antibody (mAb1) so that these antibodies could attach to the Protein G layer throuGh their nonantiGenic reGions, leavinG antiGen bindinG sites available with minimal steric hindrance for bindinG of tarGet analyte. A comparative study between this method and the more conventional strateGy of covalently attachinG a layer of nonthiolated Protein G on an alkanethiol self-assembled monolayer-coated Gold electrode has been performed. Based on a reduced preparation time, and an enhanced capac...
Jeong-woo Choi - One of the best experts on this subject based on the ideXlab platform.
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The development of Protein chip usinG Protein G for the simultaneous detection of various pathoGens
Ultramicroscopy, 2008Co-Authors: Jeong-woo Choi, Young-kee KimAbstract:Abstract A Protein chip usinG Protein G for the simultaneous detection of various pathoGens such as Escherichia coli O157:H7, Salmonella typhimurium , Yersinia enterocolitica , and LeGionella pneumophila was developed. In order to endow the orientation of antibody molecules on solid surface, Protein G was introduced. The Protein G on Gold (Au) surface modified with 11-mercaptoundecanoic acid (MUA) was arrayed and then four different kinds of monoclonal antibodies (Mabs) aGainst pathoGens ( E. coli O157:H7, S. typhimurium , Y. enterocolitica , and L. pneumophila ) on Protein G spots were selectively arrayed usinG a microarrayer, and its spatial density was over 2400 spots cm 2 . UsinG the constructed Protein chip, the various pathoGens such as E. coli O157:H7, S. typhimurium , Y. enterocolitica , and L. pneumophila could be detected by a sandwich method and its lowest detection limit for E. coli O157:H7 was 10 2 CFU/ml. The proposed fabrication technique of Protein chip for the detection of various pathoGens could be applied to construct other Protein chips with a hiGh efficiency.
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Analysis of direct immobilized recombinant Protein G on a Gold surface.
Ultramicroscopy, 2008Co-Authors: Hyunhee Kim, Da-yeon Kang, Hyun-jeong Goh, Ravindra P. Singh, Jeong-woo ChoiAbstract:For the immobilization of IGG, various techniques such as chemical linker, thiolated Protein G methods, and fraGmentation of antibodies have been reported [Y.M. Bae, B.K. Oh, W. Lee, W.H. Lee, J.W. Choi, Biosensors Bioelectron. 21 (2005) 103; W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Colloids Surf. B-Biointerfaces, 40 (2005) 143; A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. EGorov, Anal. Chem. 72 (2000) 3805]. Here, we modified the immunoGlobulin Fc-bindinG B-domain of Protein G to contain two cysteine residues at its C-terminus by a Genetic enGineerinG technique. The resultinG recombinant Protein, RPGcys, retained IGG-bindinG activity in the same manner as native Protein G. RPGcys was immobilized on a Gold surface by stronG affinity between thiol of cysteine and Gold. The orientations of both IGG layers immobilized on the base recombinant Protein Gs were analyzed by fluorescence microscope, atomic force microscope (AFM), and surface plasmon resonance (SPR). Our data revealed that IGG-bindinG activity of RPGcys on Gold surface siGnificantly increased in comparison to wild type of Protein G (RPGwild), which was physically adsorbed due to absence of cysteine residue. Immobilization of hiGhly oriented antibodies based on cysteine-modified Protein G could be useful for the fabrication of immunosensor systems.
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Study on orientation of immunoGlobulin G on Protein G layer
Biosensors & bioelectronics, 2005Co-Authors: Young Min Bae, Woochang Lee, Won Hong Lee, Jeong-woo ChoiAbstract:A comparative study of immunoGlobulin G (IGG) immobilization was performed, both on a thiolated Protein G layer, where this immobilization was due to affinity bindinG with an Fc fraGment of IGG, and on 11-mercaptoundecanoic acid (11-MUA), where the immobilization was due to chemical bondinG. The chanGe of IGG layer formation on the two base layers as a function of the IGG concentration was investiGated by surface plasmon resonance (SPR), atomic force microscopy (AFM) in a non-contact mode, and spectroscopic ellipsometry (SE). It was observed that the IGG layer was immobilized more evenly on the thiolated Protein G layer than on the 11-MUA layer, based on the SPR measurements. The surface topoloGy analysis by AFM indicated that the IGG layer was immobilized on the Protein G layer accordinG to the envelope profile of the base layer. Based on the SE analysis, it was determined that the IGG layer thickness on the thiolated Protein G layer increased with increasinG IGG concentration. Based on the above analyses, the scheme for orientation of IGG immobilized on the thiolated Protein G layer was proposed.
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Surface plasmon resonance immunosensor usinG self-assembled Protein G for the detection of Salmonella paratyphi.
Journal of biotechnology, 2004Co-Authors: Woochang Lee, Won Hong Lee, Young-kee Kim, Jeong-woo ChoiAbstract:A surface plasmon resonance (SPR) based immunosensor usinG self-assembled Protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate bindinG affinity to Protein G, the free amine (-NH2) of Protein G was substituted into thiol (-SH) usinG 2-iminothiolane. Thus, self-assembled Protein G was fabricated on Gold (Au) substrate. The formation of Protein G layer on Au surface, and the bindinG of antibody and antiGen in series were confirmed by SPR spectroscopy. The surface morpholoGy analysis of the Protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi usinG self-assembled Protein G was developed with a detection ranGe of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or Protein chips.
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Fabrication of Protein G LB film for immunoGlobulin G immobilization
Materials Science and Engineering: C, 2003Co-Authors: Bum Suk Chun, Kwang-won Park, Woochang Lee, Won Hong Lee, Jeong-woo ChoiAbstract:Abstract For the development of an immunosensor and Protein chip with hiGh efficiency, the thin film of Protein G to has specific affinity to the Fc portion of immunoGlobulin G (IGG) was fabricated by usinG the LanGmuir–BlodGett (LB) technique. The formation of Protein G LB film on a solid surface was confirmed by surface plasmon resonance (SPR), and the surface morpholoGies of the Protein G LB film were observed by usinG atomic force microscopy (AFM). The IGG bindinG to Protein G LB film and antiGen bindinG to immobilized IGG were verified by SPR measurement.
Hongcheng Liu - One of the best experts on this subject based on the ideXlab platform.
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Effect of the conserved oliGosaccharides of recombinant monoclonal antibodies on the separation by Protein A and Protein G chromatoGraphy
Journal of chromatography. A, 2009Co-Authors: Georgeen Gaza-bulseco, Keith Hickman, Sara Sinicropi-yao, Karen Hurkmans, Chris Chumsae, Hongcheng LiuAbstract:Glycosylation of the conserved asparaGine residue in CH2 domains of IGG molecules is an important post-translational modification. The presence of oliGosaccharides is critical for structure, stability and bioloGical function of IGG antibodies. Effect of the Glycosylation states of recombinant monoclonal antibodies on Protein A and Protein G chromatoGraphy was evaluated. Antibodies lackinG oliGosaccharides eluted later from Protein A and earlier from Protein G columns than antibodies with oliGosaccharides usinG a Gradient of decreasinG pH. InterestinGly, different types of oliGosaccharides also affected the elution of the antibodies. Antibodies with hiGh mannose type oliGosaccharides were enriched in later elutinG fractions from Protein A and earlier elutinG fractions from Protein G. While antibodies with more mature oliGosaccharides, such as core fucosylated biantennary complex oliGosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal Galactoses, were enriched in earlier elutinG fractions from Protein A and in the later elutinG fractions from Protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody bindinG affinity to Protein A and Protein G was not affected by the absence or presence of oliGosaccharides. It was thus concluded that the elution difference of antibodies with or without oliGosaccharides and antibodies with different types of oliGosaccharides were due to differential structural chanGes around the CH2-CH3 domain interface under the low pH conditions used for Protein A and Protein G chromatoGraphy.
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Effect of methionine oxidation of a recombinant monoclonal antibody on the bindinG affinity to Protein A and Protein G
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2008Co-Authors: Georgeen Gaza-bulseco, Karen Hurkmans, Chris Chumsae, Sagar Faldu, Hongcheng LiuAbstract:Oxidation of methionine (Met) residues is one of the most common Protein deGradation pathways. Two Met residues, Met256 and Met432, of a recombinant fully human monoclonal IGG1 antibody have been shown to be susceptible to oxidation. Met256 and Met432 are located in the antibody CH2-CH3 interface and in close proximity to Protein A and Protein G bindinG sites. The effect of oxidation of these susceptible Met residues on the bindinG to Protein A and Protein G was investiGated in the current study. Incubation of the antibody with 5% tert-butyl hydroperoxide (tBHP) resulted in a nearly complete oxidation of Met256 and Met432, while incubation with 1% tBHP resulted in mixed populations of the antibody with different deGrees of Met oxidation. Oxidation of Met256 and Met432 resulted in earlier elution of the antibody from Protein A and Protein G columns when eluted with a Gradient of decreasinG pH. Analysis by ELISA and surface plasmon resonance (SPR) revealed decreased bindinG affinity of the oxidized antibody to Protein A and Protein G. It is therefore concluded that oxidation of the Met256 and Met432 residues of the recombinant monoclonal antibody altered its interaction with Protein A and Protein G resultinG in a decrease in bindinG affinity.
Georgeen Gaza-bulseco - One of the best experts on this subject based on the ideXlab platform.
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Effect of the conserved oliGosaccharides of recombinant monoclonal antibodies on the separation by Protein A and Protein G chromatoGraphy
Journal of chromatography. A, 2009Co-Authors: Georgeen Gaza-bulseco, Keith Hickman, Sara Sinicropi-yao, Karen Hurkmans, Chris Chumsae, Hongcheng LiuAbstract:Glycosylation of the conserved asparaGine residue in CH2 domains of IGG molecules is an important post-translational modification. The presence of oliGosaccharides is critical for structure, stability and bioloGical function of IGG antibodies. Effect of the Glycosylation states of recombinant monoclonal antibodies on Protein A and Protein G chromatoGraphy was evaluated. Antibodies lackinG oliGosaccharides eluted later from Protein A and earlier from Protein G columns than antibodies with oliGosaccharides usinG a Gradient of decreasinG pH. InterestinGly, different types of oliGosaccharides also affected the elution of the antibodies. Antibodies with hiGh mannose type oliGosaccharides were enriched in later elutinG fractions from Protein A and earlier elutinG fractions from Protein G. While antibodies with more mature oliGosaccharides, such as core fucosylated biantennary complex oliGosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal Galactoses, were enriched in earlier elutinG fractions from Protein A and in the later elutinG fractions from Protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody bindinG affinity to Protein A and Protein G was not affected by the absence or presence of oliGosaccharides. It was thus concluded that the elution difference of antibodies with or without oliGosaccharides and antibodies with different types of oliGosaccharides were due to differential structural chanGes around the CH2-CH3 domain interface under the low pH conditions used for Protein A and Protein G chromatoGraphy.
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Effect of methionine oxidation of a recombinant monoclonal antibody on the bindinG affinity to Protein A and Protein G
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2008Co-Authors: Georgeen Gaza-bulseco, Karen Hurkmans, Chris Chumsae, Sagar Faldu, Hongcheng LiuAbstract:Oxidation of methionine (Met) residues is one of the most common Protein deGradation pathways. Two Met residues, Met256 and Met432, of a recombinant fully human monoclonal IGG1 antibody have been shown to be susceptible to oxidation. Met256 and Met432 are located in the antibody CH2-CH3 interface and in close proximity to Protein A and Protein G bindinG sites. The effect of oxidation of these susceptible Met residues on the bindinG to Protein A and Protein G was investiGated in the current study. Incubation of the antibody with 5% tert-butyl hydroperoxide (tBHP) resulted in a nearly complete oxidation of Met256 and Met432, while incubation with 1% tBHP resulted in mixed populations of the antibody with different deGrees of Met oxidation. Oxidation of Met256 and Met432 resulted in earlier elution of the antibody from Protein A and Protein G columns when eluted with a Gradient of decreasinG pH. Analysis by ELISA and surface plasmon resonance (SPR) revealed decreased bindinG affinity of the oxidized antibody to Protein A and Protein G. It is therefore concluded that oxidation of the Met256 and Met432 residues of the recombinant monoclonal antibody altered its interaction with Protein A and Protein G resultinG in a decrease in bindinG affinity.
Jeremy P. Derrick - One of the best experts on this subject based on the ideXlab platform.
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Use of streptococcal Protein G in obtaininG crystals of an antibody Fab fraGment in complex with a meninGococcal antiGen.
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Jeremy P. Derrick, Ian M. Feavers, Martin C. J. MaidenAbstract:Crystals have been obtained of an antibody Fab fraGment Grown in the presence of a sinGle domain from streptococcal Protein G and a ten amino-acid peptide correspondinG to the P1.7 serosubtype antiGen from the human pathoGen Neisseria meninGitidis. Crystal trials usinG the Fab fraGment and peptide antiGen alone were unsuccessful, but the inclusion of a Protein G domain provided an additional variable that Generated suitable crystals. Crystals are in space Group P21 with unit-cell parameters a = 43.60, b = 63.42, c = 89.63 A, β = 98.58° and a data set has been collected to 2.9 A resolution usinG synchrotron radiation. The inclusion of Protein G is likely to be of General utility for the crystallization of Fab–antiGen complexes.
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Research ArticleModel for the complex between Protein G and an antibody Fc fraGment in solution
1995Co-Authors: Koichi Kato, Jeremy P. Derrick, Lu-yun Lian, Igor L. Barsukov, Runa Tanaka, Atsuko Yoshino, Miki Shiraishi, Ichio Shimada, Yoji ArataAbstract:BackGround: Streptococcal Protein G and staphylococcal Protein A are bacterial antibody-bindinG Proteins, widely used as immunoloGical tools, whose antibody-bindinG domains are structurally quite different. The bindinG of Protein G to Fc fraGments is competitive with respect to Protein A, suGGestinG that the bindinG sites for Protein A and Protein G on Fc overlap, notwithstandinG the fact that they lack sequence or structural similarity. . Results To resolve this issue, the residues involved in the interaction between an IGG-bindinG domain of Protein G (domain II) and the Fc fraGment of mouse IGG2a have been identified by use of 13 C and 15 N NMR. BindinG of Protein G domain II selectively perturbed resonances from residues between the CH 2 and CH 3 domains of Fc, whereas in domain II the residues affected are primarily those on the α–helix and the third strand of the β–sheet. This information was used, toGether with the structures of the two uncomplexed Proteins, to construct a model of the complex, usinG Monte Carlo minimization techniques. In this model, the α–helix of Protein G lies in the same position as helix 1 of Protein A in the crystal structure of the Protein A:Fc complex, but its orientation differs from the latter by 180°. . Conclusion The interactions of the bacterial antibody-bindinG Proteins with their ‘tarGet’ immunoGlobulins involve a very versatile set of Protein–Protein interactions. First, the IGG-bindinG domains of Protein A and Protein G have quite different three-dimensional structures, but bind to sites on the Fc fraGment that overlap extensively. Secondly, Protein G employs two quite different reGions of its surface to bind to the Fab and Fc reGions of IGG.
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Model for the complex between Protein G and an antibody Fc fraGment in solution
Structure, 1995Co-Authors: Koichi Kato, Jeremy P. Derrick, Lu-yun Lian, Igor L. Barsukov, Runa Tanaka, Atsuko Yoshino, Miki Shiraishi, Ichio Shimada, Yoji ArataAbstract:Abstract BackGround: Streptococcal Protein G and staphylococcal Protein A are bacterial antibody-bindinG Proteins, widely used as immunoloGical tools, whose antibody-bindinG domains are structurally quite different. The bindinG of Protein G to Fc fraGments is competitive with respect to Protein A, suGGestinG that the bindinG sites for Protein A and Protein G on Fc overlap, notwithstandinG the fact that they lack sequence or structural similarity. . Results To resolve this issue, the residues involved in the interaction between an IGG-bindinG domain of Protein G (domain II) and the Fc fraGment of mouse IGG2a have been identified by use of 13 C and 15 N NMR. BindinG of Protein G domain II selectively perturbed resonances from residues between the C H 2 and C H 3 domains of Fc, whereas in domain II the residues affected are primarily those on the α –helix and the third strand of the β –sheet. This information was used, toGether with the structures of the two uncomplexed Proteins, to construct a model of the complex, usinG Monte Carlo minimization techniques. In this model, the α –helix of Protein G lies in the same position as helix 1 of Protein A in the crystal structure of the Protein A:Fc complex, but its orientation differs from the latter by 180°. . Conclusion The interactions of the bacterial antibody-bindinG Proteins with their ‘tarGet' immunoGlobulins involve a very versatile set of Protein–Protein interactions. First, the IGG-bindinG domains of Protein A and Protein G have quite different three-dimensional structures, but bind to sites on the Fc fraGment that overlap extensively. Secondly, Protein G employs two quite different reGions of its surface to bind to the Fab and Fc reGions of IGG.
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Crystal structure of a streptococcal Protein G domain bound to an Fab fraGment.
Nature, 1992Co-Authors: Jeremy P. Derrick, Dale B. WigleyAbstract:Protein G is a cell-surface Protein from Streptococcus1,2 which binds to IGG molecules from a wide ranGe of species with an affinity comparable to that of antiGen3,4. The hiGh affinity of Protein G for the Fab portion of IGG poses a particular challenGe in molecular recoGnition, Given the variability of heavy chain subclass, liGht chain type and complementarity-determininG reGions. Here we report the crystal structure of a complex between a Protein G domain and an immunoGlobulin Fab fraGment. An outer β-strand in the Protein G domain forms an antiparallel interaction with the last β-strand in the constant heavy chain domain of the immunoGlobulin, thus extendinG the β-sheet into the Protein G. The interaction between secondary structural elements in Fab and Protein G provides an inGenious solution to the problem of maintaininG a hiGh affinity for many different IGG molecules. The structure also contrasts with Fab–antiGen complexes, in which all contacts with antiGen are mediated by the variable reGions of the antibody, and to our knowledGe provides the first details of interaction of the constant reGions of Fab with another Protein.
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Crystal structure of a streptococcal Protein G domain bound to an Fab fraGment.
Nature, 1992Co-Authors: Jeremy P. Derrick, Dale B. WigleyAbstract:Protein G is a cell-surface Protein from Streptococcus which binds to IGG molecules from a wide ranGe of species with an affinity comparable to that of antiGen. The hiGh affinity of Protein G for the Fab portion of IGG poses a particular challenGe in molecular recoGnition, Given the variability of heavy chain subclass, liGht chain type and complementarity-determininG reGions. Here we report the crystal structure of a complex between a Protein G domain and an immunoGlobulin Fab fraGment. An outer beta-strand in the Protein G domain forms an antiparallel interaction with the last beta-strand in the constant heavy chain domain of the immunoGlobulin, thus extendinG the beta-sheet into the Protein G. The interaction between secondary structural elements in Fab and Protein G provides an inGenious solution to the problem of maintaininG a hiGh affinity for many different IGG molecules. The structure also contrasts with Fab-antiGen complexes, in which all contacts with antiGen are mediated by the variable reGions of the antibody, and to our knowledGe provides the first details of interaction of the constant reGions of Fab with another Protein.
