Academic literature on the topic 'Recombinant proteins Gold Protein binding'
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Journal articles on the topic "Recombinant proteins Gold Protein binding"
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Breitwieser, Andreas, Philipp Siedlaczek, Helga Lichtenegger, Uwe B. Sleytr, and Dietmar Pum. "S-Layer Protein Coated Carbon Nanotubes." Coatings 9, no. 8 (August 2, 2019): 492. http://dx.doi.org/10.3390/coatings9080492.
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Carbon nanotubes (CNTs) have already been considered for medical applications due to their small diameter and ability to penetrate cells and tissues. However, since CNTs are chemically inert and non-dispersible in water, they have to be chemically functionalized or coated with biomolecules to carry payloads or interact with the environment. Proteins, although often only randomly bound to the CNT surface, are preferred because they provide a better biocompatibility and present functional groups for binding additional molecules. A new approach to functionalize CNTs with a closed and precisely ordered protein layer is offered by bacterial surface layer (S-layer) proteins, which have already attracted much attention in the functionalization of surfaces. We could demonstrate that bacterial S-layer proteins (SbpA of Lysinibacillus sphaericus CCM 2177 and the recombinant fusion protein rSbpA31-1068GG comprising the S-layer protein and two copies of the IgG binding region of Protein G) can be used to disperse and functionalize oxidized multi walled CNTs. Following a simple protocol, a complete surface coverage with a long-range crystalline S-layer lattice can be obtained. When rSbpA31-1068GG was used for coating, the introduced functionality could be confirmed by binding gold labeled antibodies via the IgG binding domain of the fusion protein. Since a great variety of functional S-layer fusion proteins has already been described, our new technology has the potential for a broad spectrum of functionalized CNTs.
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Frevert, U., S. Sinnis, C. Cerami, and V. Nussenzweig. "Localization of cell-surface and basement-membrane heparan-sulfate proteoglycans using the malaria circumsporozoite protein." Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 294–95. http://dx.doi.org/10.1017/s0424820100169201.
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Malaria sporozoites, which invade hepatocytes within minutes after transmission by an infected mosquito, are covered with the circumsporozoite (CS) protein, which in all Plasmodium species contains the conserved region II-plus. This region is also found as a cell-adhesive motif in a variety of host proteins like thrombospondin, properdin and the terminal complement components.The CS protein with its region II-plus specifically binds to heparan sulfate proteoglycans (HSPG) on the basolateral surface of hepatocytes in the space of Disse (FIG. 1), to certain basolateral cell membranes and basement membranes of the kidney (FIG. 2) as well as to heparin in the granules of connective tissue mast cells. The distribution of the HSPG receptors for the CS protein was examined by incubation of Lowicryl K4M or LR White sections of liver and kidney tissue with the recombinant CS ligand, whose binding sites were detected with a monoclonal anti-CS antibody and protein A gold.
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Ghitescu, L., Z. Galis, and M. Bendayan. "Protein AG-gold complex: an alternative probe in immunocytochemistry." Journal of Histochemistry & Cytochemistry 39, no. 8 (August 1991): 1057–65. http://dx.doi.org/10.1177/39.8.1856455.
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The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.3, which displays immunoglobulin Fc binding sites for both staphylococcal protein A and streptococcal protein G. It adsorbs to 10-nm colloidal gold particles with a lower affinity than does protein A, and is saturable. A maximal number of 12 protein AG molecules could be accommodated on the gold particle surface. Protein AG-gold conjugates yielded positive signals in post-embedding immunocytochemical assays when used as a secondary reagent in conjunction with several species and classes of polyclonal (rabbit, goat, sheep, guinea pig) and mouse monoclonal immunoglobulins (IgG1, IgG2, and IgG3). In addition, protein AG-gold was found to be a useful reagent in immunoblot analysis because of its ability to bind and identify nitrocellulose-immobilized IgGs (rabbit, mouse, goat, sheep, rat, and cow). Its spectrum of specificity towards various types of antibodies combines those of the parental protein A and protein G molecules. The protein AG-gold complex therefore appears to be a highly versatile and convenient alternative probe for immunochemical and immunocytochemical studies.
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Rezniczek, Günther A., José M. de Pereda, Siegfried Reipert, and Gerhard Wiche. "Linking Integrin α6β4-based Cell Adhesion to the Intermediate Filament Cytoskeleton: Direct Interaction between the β4 Subunit and Plectin at Multiple Molecular Sites." Journal of Cell Biology 141, no. 1 (April 6, 1998): 209–25. http://dx.doi.org/10.1083/jcb.141.1.209.
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Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin's interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the β4 subunit of the basement membrane laminin receptor integrin α6β4 that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin β4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin β4 subunits and cytokeratin filaments.
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Molinková, D. "Purification of Escherichia coli-expressed HIS-tagged Maedi-Visna p25 core antigen by Ni2+-chelate affinity chromatography ." Veterinární Medicína 46, No. 2 (January 1, 2001): 50–54. http://dx.doi.org/10.17221/7852-vetmed.
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In this study, recombinant histidine tagged p25 capsid protein of Maedi-Visna virus was developed. Part of the viral genome coding p25 protein was positioned downstream and in frame with a metal binding domain in pRSET-B vector. Recombinant protein was expressed in E. coli cells and soluble fraction of the protein was subsequently purified by Ni2+-chelate affinity chromatography. Purified protein was then used as antigen in an indirect ELISA test. One hundred fifty ovine serum samples were screened for antibodies to p25 protein of the virus. Immunoblot with whole virus antigen was used as a gold standard. The total number of positive results in the ELISA was 38 (25.33%). Immunoblot failed to confirm a positive result in 2 (1.33%) of them and these results were therefore considered to be false positive. The number of true positive results in the ELISA was thus 36 (24%). All immunoblot positive samples were also positive by ELISA test. In conclusion, recombinant His-tagged capsid protein showed very high sensitivity and specificity in detecting antibodies to Maedi-Visna virus.
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GOBERT, G. N., D. J. STENZEL, M. K. JONES, and D. P. McMANUS. "Immunolocalization of the fatty acid-binding protein Sj-FABPc within adult Schistosoma japonicum." Parasitology 115, no. 1 (July 1997): 33–39. http://dx.doi.org/10.1017/s0031182097008925.
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This paper describes the first localization study of the 14·7 kDa fatty acid-binding protein in Schistosoma japonicum (Sj-FABPc) using transmission electron microscopy. A polyclonal antibody raised against recombinant Sj-FABPc was used in combination with a colloidal gold marker to determine the distribution of the protein within adult parasites. Sj-FABPc was localized within lipid droplets below the subtegumental region of the male parasite. Additionally, Sj-FABPc was present in the vitelline droplets of the vitelline glands of female parasites. There were no detectable levels of Sj-FABPc on the surface or within the tegument of male or female parasites. Possible functions of Sj-FABPc within S. japonicum and the relevance of these immunolocalization findings in light of the recent reports that the homologue Sm-FABPc is an important anti-S. mansoni vaccine target molecule are also discussed.
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Preobrazhenskaya, Y. V., A. I. Stenko, M. V. Shvarts, and V. Y. Lugovtsev. "Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP." Journal of Amino Acids 2013 (January 30, 2013): 1–8. http://dx.doi.org/10.1155/2013/983565.
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The transformation of the strain DH5αTM-T1R with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E. coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA. 28 mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with KDs different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit.
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Matsui, Hidehito, Hideaki Hanaki, Megumi Inoue, Hiroyuki Akama, Taiji Nakae, Keisuke Sunakawa, and Satoshi Omura. "Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′." Clinical and Vaccine Immunology 18, no. 2 (December 22, 2010): 248–53. http://dx.doi.org/10.1128/cvi.00252-10.
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ABSTRACTInfections with methicillin-resistantStaphylococcus aureus(MRSA) and methicillin-resistant coagulase-negativeStaphylococcus(MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2′ (PBP2′ or PBP2a), which shows low affinity to β-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2′ within about 20 min. Anti-PBP2′ monoclonal antibodies were produced by a hybridoma of recombinant PBP2′ (rPBP2′)-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2′ of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2′, and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2′-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2′ or 2.8 × 105to 1.7 × 107CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and aCandida albicansstrain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2′ gene,mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2′-producing cells in clinical laboratories.
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Heo, Yunseok, Hyeongseop Jeong, Youngki Yoo, Ji-Hye Yun, Bumhan Ryu, Young-je Cha, Bo-Ram Lee, et al. "Structural and Functional Characterizations of Cancer Targeting Nanoparticles Based on Hepatitis B Virus Capsid." International Journal of Molecular Sciences 22, no. 17 (August 24, 2021): 9140. http://dx.doi.org/10.3390/ijms22179140.
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Cancer targeting nanoparticles have been extensively studied, but stable and applicable agents have yet to be developed. Here, we report stable nanoparticles based on hepatitis B core antigen (HBcAg) for cancer therapy. HBcAg monomers assemble into spherical capsids of 180 or 240 subunits. HBcAg was engineered to present an affibody for binding to human epidermal growth factor receptor 1 (EGFR) and to present histidine and tyrosine tags for binding to gold ions. The HBcAg engineered to present affibody and tags (HAF) bound specifically to EGFR and exterminated the EGFR-overexpressing adenocarcinomas under alternating magnetic field (AMF) after binding with gold ions. Using cryogenic electron microscopy (cryo-EM), we obtained the molecular structures of recombinant HAF and found that the overall structure of HAF was the same as that of HBcAg, except with the affibody on the spike. Therefore, HAF is viable for cancer therapy with the advantage of maintaining a stable capsid form. If the affibody in HAF is replaced with a specific sequence to bind to another targetable disease protein, the nanoparticles can be used for drug development over a wide spectrum.
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Gao, Jinlong, Daniele Vicari, Nan Li, and Charles Collyer. "Type II cleaved gingipain adhesins act in bacterial invasion." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C807. http://dx.doi.org/10.1107/s205327331409192x.
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Gingipains are multi domain peptidases that are primary virulence factors expressed by the keystone periodontal disease pathogen P. gingivalis. The adhesion regions of these proteases are multi-domain complexes that are comprised of a number of modules that belong to the type 1 (T1) family of gingipain adhesins (also known as cleaved adhesin domains). We have to date reported the crystal structures of three homologous variants of these 19 kDa T1 domains and shown that they recognise a number of host target proteins. We have proceeeded to predict from sequence analysis and binding data that in addition to the T1 domains the adhesion regions also contain a number of other structurally (and sequence) unrelated adhesins (referred to here as type 2 or T2 gingpain adhesins) that may synergistically contribute to the virulence of P. gingivalis. We have recombinantly expressed and crystallized the first example of an 17.5 kDA T2 adhesin coded for by a fragment of the gene for the lysine specific gingipain (kgp). We report here the structure refined at 1.05 angstroms resolution and thereby confirm our hypothesis of the existence of the T2 domain family. This structure represents a new fold family which is distantly similar in topology to that of the plastocyanin/azurin family of proteins but with no copper binding sites. Like the T1 adhesins it contains a structural binding site for calcium. We observe that the recombinant T2 adhesin found in the gingipain Kgp also binds directly to host target proteins but more importantly can also competitively inhibit a bacterial invasion cell based assay. The role of this new T2 adhesin in a specific adhesion activity in the cell invasion mechanism is likely a potential critical component in the overall virulence of this pathogen. Figure. Cartoon representation of the superposition of the structures of the Kgp T2 adhesin (green cartoon and blue calcium) and azurin (pink cartoon and gold copper - pdb code 3NP3)
Dissertations / Theses on the topic "Recombinant proteins Gold Protein binding"
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Suzuki, Noriaki. "Applications of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and x-ray photoelectron spectroscopy (XPS) to study interactions of genetically engineered proteins with noble metal films /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/10618.
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Tse, Muk-hei. "Investigations on recombinant Arabidopsis acyl-coenzyme A binding protein 1." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36427664.
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Tse, Muk-hei, and 謝牧熙. "Investigations on recombinant Arabidopsis acyl-coenzyme A binding protein 1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B36427664.
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Vrable, David Joseph. "A recombinant GST-EMBP440 fusion protein from sea urchin embryos that has myosin binding capabilities /." Connect to online version, 1997. http://hdl.handle.net/1989/3741.
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Ndabambi, Nonkululeko. "Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies." Thesis, University of the Western Cape, 2004. http://etd.uwc.ac.za/index.php?module=etd&.
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The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.
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Lundell, Emma. "Interactions of the Human Recombinant Proteins JUNO and IZUMO1." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-277123.
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Det uppskattas att 15% av alla par världen över lider avinfertilitet. Ungefär hälften beror på manlig infertilitet och 40% av dessa fall kan ännu inte förklaras. Därmed är nuvarande metoder för att diagnostisera manlig infertilitet otillräckliga och ytterligare tekniker behövs. En lyckad befruktning kräver att spermierna uttrycker membranproteinet Izumo1 som måste känna igen dess receptorprotein Juno, belägen vid ytan av äggmembranet. Bindningen mellan Juno och Izumo1 är essentiell för befruktning hos däggdjur då den bidrar till att gameternabinder och skapar en ny distinkt organism. Juno är ett relativt nyupptäckt protein och mekanismen med Izumo1 är fortfarande okänd. Ett nystartat företag vid namn Spermosens vill mäta interaktionen mellan Juno ochIzumo1 i ett nytt diagnostikverktyg som ska diagnostisera manlig infertilitet. Tanken är att Juno ska immobiliseras på guld-nanopartiklar och användas för att mätainteraktionen med spermaprover. Det nya verktyget ska hjälpa par att fastställa felet i befruktningen, vilket som en följd skulle hjälpa paret att välja lämplig assisteradreproduktionsmetod. I utvecklingen av det nya diagnostikverktyget behöver det konfirmeras att den Juno som används i enheten kan binda korrekt till mänskligt Izumo1. Därför måste interaktionerna mellan de mänskliga, rekombinanta proteinerna Juno och Izumo1 mätas och karakteriseras. Syftet med detta projekt var att utveckla en metod för att immobilisera Juno på guldnanopartiklar och sedan mäta interaktionerna med Izumo1 genom UV-Visspektroskopi. Detta är teoretiskt möjligt eftersom guld-nanopartiklarna framkallar ett fenomen som kallas lokaliserad ytplasmonresonans som varierar beroende påstorleken på guld-nanopartikelkomplexet. Immobiliseringsmetoden var en process som involverade flera steg som designades, polerades och förbättrades underarbetets gång. Dithiobis(C2-NTA) konjugerades till guldytan och koboltjonerkonjugerades till NTA. Det sista steget som innebar konjugering av Juno till kobolt genom en His-tag lyckades inte, och interaktionerna kunde därför inte mätas genom denna metod. Istället mättes protein-protein-interaktionen genom SPR-mätningar med Biacore, ett instrument som också är baserat på ytplasmonresonans. Interaktioner mellan Izumo1 och Juno kunde uppmätas både vid användning av Juno producerad från E. coli ochfrån däggdjursceller. Dissociationskonstanten (Kd) beräknades till 7-33 nM (för Junooch Izumo1 producerade i däggdjursceller) vilket kan jämföras med ett experimentfrån 2016 där 48 nM beräknades. Ett mer exakt Kd kunde inte fastställas och entrolig anledning till detta var att regenereringen av sensorytan som utfördes med NaOH varierade i effektivitet, vilket ledde till en osäkerhet då ytförhållandena kan ha varierat mellan mätningarna. De två Juno-proteinerna, som är producerade i olika organismer, visade två skilda affinitetsprofiler med Izumo1 vilket tyder på att glykosyleringen påverkar bindningsmekanismen mellan Juno och Izumo1.It is estimated that 15% of all couples worldwide suffer from infertility. Roughly half is male-factor infertility and 40% of these cases cannot be explained. Thus, current methods for diagnosing male infertility are not enough and further techniques are needed. To have a successful fertilisation event, it is required that the sperm expresses membrane surface-protein Izumo1 which must recognise its counterpart protein Juno, located at the surface of the egg membrane. The recognition step between Juno and Izumo1 is essential in mammalian fertilisation for the gametes to bind and start the creation of a new distinct organism, but the molecular mechanism is still unknown.A start-up company named Spermosens want to measure the Juno-Izumo1 interaction in a new diagnostic device designed to diagnose male infertility. The idea is to have Juno immobilised on gold nanoparticles and measure the interaction between Juno and various semen samples. The new device is supposed to help couples pin-point the procreation issue which would help in the selection of suitable assisted reproductive technology. In the development of the new device, it had to be established that the Juno used in the device will bind correctly to human Izumo1. Therefore, the interactions between the human recombinant proteins Juno and Izumo1 had to be measured and characterized.The objectives of this project were to develop a method to immobilise Juno on gold nanoparticles and then measure the interactions with Izumo1 using UV-vis spectroscopy. This is theoretically possible since the gold nanoparticles exhibit a phenomenon called localized surface plasmon resonance that vary depending on the size of the gold nanoparticle-complex. The immobilisation procedure was a process involving several steps that were designed, polished and improved along the way. Dithiobis(C2-NTA) was conjugated to the gold surface and a cobalt ion was conjugated to the NTA. The last step involving conjugation of Juno to the cobalt through a His-tag was not succeeded, and the interactions could therefore not be measured this way.Instead, the protein-protein interaction was measured through SPR-measurements using Biacore, an instrument that is based on surface plasmon resonance as well. Interactions between Izumo1 and Juno could be detected using Juno produced in E. coli and in mammalian cells. The dissociation constant (Kd) could be calculated to 7-33 nM which can be compared to a previously published Kd of 48 nM. A more precise Kd could not be established, possibly due to that the regeneration of the sensor surface with NaOH varied in efficiency, leading to changing surface conditions during the measurements. The two Juno proteins, that were produced in different hosts, showed two different affinity profiles with Izumo1, which contributes to the suggestion that the glycosylation plays a role in the binding mechanism between Juno and Izumo1.
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Kepple, Kevin V. "Analysis of the binding mechanisms and cellular targets of peptide inhibitors that block site-specific recombination in vitro /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3208620.
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Moore, Shona. "An analysis of the structure and function of malarial Duffy-binding-like protein domains using recombinant fusion proteins." Thesis, University of Warwick, 2016. http://wrap.warwick.ac.uk/86933/.
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Duffy-binding-like domains are present in two potential malaria vaccine candidates. Located on the merozoite surface, MSPDBL1 and MSPDBL2 have been implicated in erythrocyte invasion and identified as targets of natural immunity. Merozoite DBL domains have been shown to bind the Fc region of natural IgM. This is characteristic of several PfEMP1s, and is also well documented in bacteria, viruses and other parasites, where it is thought to prevent specific binding of the more deadly IgG antibodies. We have developed a mammalian expression system to produce merozoite DBL domains as Fc fusion proteins, facilitating investigation into their adhesive properties. Fc-fusion proteins are composed of the Fc region of IgG fused to a peptide and are a rapidly expanding field of bio-engineering. They have been successful in drug delivery due to their ability to increase serum half-life of the fused protein by the interaction of the IgG Fc with the neonatal Fc receptor (FcRn). Engineering of the Fc scaffold has shown improved receptor binding, allowing cross-linking of Fc receptors for improved vaccine design. The expression of homodimeric DBL-Fc fusions is di cult, evidenced by incorrect folding and low protein yield. A flexible ,extended hinge region was designed to increase the distance between the Fc and the fused DBL domain, and improved protein folding and IgM binding. Further work may optimise this hinge region for the development of malarial vaccines, or therapeutics for IgM-mediated diseases. The structural analysis of all known IgM-binding DBL domains and residues on the merozoite DBL surface predict the involvement of helix 2a in IgM binding. This contradicts a recent homology model of the IgM-binding interaction, and suggests that the model needs revision. An improved DBL-Fc fusion could be used to identify critical binding residues located in this helix using the more focused approach of site-directed mutagenesis.
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Levchenko, Tetyana. "The role of angiomotin in angiogenesis /." Stockholm, 2003. http://diss.kib.ki.se/2004/91-7349-761-4/.
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Greenham, Trevor. "Pointillism in Plant Systems Biology: I. Proteomic Analysis of Plant Exosome-like Particles II. Amyloplast-binding Puroindoline Fusion Proteins for Recombinant Protein Expression." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39647.
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Expanding upon our understanding of plant defense is critical, particularly with the perilous threats of climate change and overpopulation to our food security, health and well-being. In this study, we focused on plant defense using two distinct approaches. First, we performed a proteomic analysis of plant exosome-like nanoparticles in order to elucidate their defense related protein cargo. Secondly, we used a wheat antimicrobial protein, puroindoline, as a fusion partner for the expression of recombinant proteins in rice endosperm.
Plant exosome-like nanoparticles (ELP) were isolated from fresh tomato and subjected to mass spectrometry (MS) analysis. The ELPs were compared to fresh pressed tomato juice, and the proteins that were significantly upregulated in the ELPs were analyzed for their defensive properties. Bioinformatic analysis identified 30 proteins upregulated in the ELPs, with a majority of these being involved in plant defense.
Puroindoline is a protein found in soft wheat varieties. A unique feature of this protein is the presence of a tryptophan-rich domain, which causes it to localize and tether onto starch granule surfaces; a property we are seeking to exploit for recombinant protein isolation. We hypothesized that when expressed in a pin-null crop, such as rice, puroindoline along with its fusion partner will localize and adhere to starch granule surfaces. PIN fusions were expressed in rice, and their subcellular localization was determined by immunolocalization. It was observed that PIN localizes to rice starch
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granules in vitro and in planta, and retains its starch granule binding abilities as a fusion partner.
To identify other possible starch granule binding fusion partners, an anhydrous cleavage method was developed that can scan dry biological materials for associated proteins, in this case the starch granule surface. Incubation of our cleavage reagent with isolated rice starch granules yielded several cleavage products as determined through SDS-PAGE. These cleavage products were compared with previous proteomic data of trypsin digested rice starch granules.
Books on the topic "Recombinant proteins Gold Protein binding"
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Hernandez, Marta. The study of ligand binding specificities of the lipid binding proteins: Recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands. St. Catharines, Ont: Brock University, Dept. of Biotechnology, 2003.
Find full textBook chapters on the topic "Recombinant proteins Gold Protein binding"
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Austin, Brian P., Sreedevi Nallamsetty, and David S. Waugh. "Hexahistidine-Tagged Maltose-Binding Protein as a Fusion Partner for the Production of Soluble Recombinant Proteins in Escherichia coli." In Methods in Molecular Biology, 157–72. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-59745-196-3_11.
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Sun, Ping, Joseph E. Tropea, and David S. Waugh. "Enhancing the Solubility of Recombinant Proteins in Escherichia coli by Using Hexahistidine-Tagged Maltose-Binding Protein as a Fusion Partner." In Methods in Molecular Biology, 259–74. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-61737-967-3_16.
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Tropea, Joseph E., Scott Cherry, Sreedevi Nallamsetty, Christophe Bignon, and David S. Waugh. "A Generic Method for the Production of Recombinant Proteins in Escherichia coli Using a Dual Hexahistidine-Maltose-Binding Protein Affinity Tag." In Methods in Molecular Biology, 1–19. Totowa, NJ: Humana Press, 2007. http://dx.doi.org/10.1007/978-1-59745-209-0_1.
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Guha, Nishan, Ioulietta Erotokritou-Mulligan, Simon P. Nevitt, Michael Francis, Christiaan Bartlett, David Cowan, E. Eryl Bassett, Peter H. Sonksen, and Richard IG Holt. "The Response of IGF-I and Pro-Collagen Type III Amino-Terminal Propeptide to the Administration of Recombinant Human IGF-I/IGF Binding Protein-3 in Recreational Athletes." In BASIC/TRANSLATIONAL - IGF & IGF Binding Proteins, P1–153—P1–153. The Endocrine Society, 2011. http://dx.doi.org/10.1210/endo-meetings.2011.part1.p6.p1-153.
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Sligar, Stephen G., and Clifford R. Robinson. "Osmotic and Hydrostatic Pressure as Tools to Study Molecular Recognition." In High Pressure Effects in Molecular Biophysics and Enzymology. Oxford University Press, 1996. http://dx.doi.org/10.1093/oso/9780195097221.003.0026.
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The question of molecular recognition is a central paradigm of molecular biology, playing central roles in most, if not all, cellular processes. Failed recognition events have been implicated in numerous disease states, ranging from flawed control of gene regulation and cellular proliferation to defects in specific metabolic activities. Historically, questions of molecular recognition have been approached through organic synthesis and through actual structural studies of biomolecular complexes. Fundamental insight into the mechanisms of molecular recognition can be realized through the use of broad interdisciplinary tools and techniques. In particular, the use of recombinant DNA technology in concert with hydrostatic and osmotic pressure methodologies have proven to be ideal for understanding the fundamental mechanisms of recognition. In our presentation, we will focus on recent results from our laboratory that examine three major classes of recognition events in biological systems: 1. Protein-protein recognition: here we seek to define the role of specific surface interactions; electrostatic, hydrogen bonding, and hydrophobic free energies provided through surface complimentarity, which define the specificity and affinity in the formation of complexes between the metalloproteins involved in electron transfer events in cytochrome P-450-dependent oxygenase catalysis and in the assembly of tetrameric hemoglobin. 2. Protein—small molecule recognition: here we seek to ascertain how the same fundamental forces of electrostatics, hydrogen bonding, and the hand-glove fit of a substrate into the active site of an enzyme can give rise to the observed high degree of control of regio- and stereo-specificity in catalysis and in the interfadal interactions of proteins at electrode interfaces. 3. Protein nucleic acid recognition: here again the same fundamental forces control recognition processes, but in this case we will focus on our exciting, recent discovery of a role for solvent water in mediating recognition between protein and nucleic acid components. Representative systems in the binding/ catalytic class of restriction endonucleases and recombinases will be discussed. In all cases, the use of pressure as a variable has provided unique understanding for the molecular details of these processes. Pressure, both hydrostatic and osmotic, has proven to be an enabling experimental technique in understanding the mechanistic origins of molecular recognition events.
Conference papers on the topic "Recombinant proteins Gold Protein binding"
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Furis, B. C., M. J. Jorgensem, M. J. Rabiet, A. B. Contor, C. L. Brown, C. B. Shoemaker, and B. Furie. "RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643992.
Full textAbstract:
Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.
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Novikova, L. I., S. S. Bochkareva, A. V. Aleshkin, S. IU Kombarova, O. E. Karpov, A. A. Pulin, O. A. Orlova, IU S. Lebedin, A. M. Vorobev, and E. R. Mekhtiev. "DYNAMICS OF ANTIBODIES TO VARIOUS ANTIGENS OF THE SARS-COV-2 CORONAVIRUS IN PATIENTS WITH CONFIRMED COVID-19 INFECTION." In Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-159.
Full textAbstract:
Presence of IgG and IgM antibodies in venous blood of 76 patients with confirmed presence of SARS-CoV-2 was determined. The study was carried out by ELISA using Russian test systems. Revealed different levels of IgM antibodies to N-protein and RBD (receptor binding domain of the Spike protein). The level of IgM to RBD did not reach high values, while the level of IgM to N-protein sharply increased in a short period of time to high values by the 3rd week of the disease and decreased only by the 8th week. The dynamics of IgG antibodies to the whole virion antigen and the recombinant spikes was similar, reaching high values at 4-5 weeks of the disease, however, the dynamics of IgG to the N-protein differed, showing a slight increase by the 1st week of the disease and a low level throughout the entire period of observation. Different dynamics of production of IgG and IgM antibodies to N-protein and RBD were noted. The amount of IgM to the N-protein increased sharply and reached a high level, while the amount of IgG increased smoothly and did not show a high level. The opposite picture was observed for antibodies to RBD. The characteristic dynamics of IgG, measured using test systems withsorbed whole virion or recombinant spike proteins, suggests duration of the disease
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Higgins, Deborah L., and William E. Holmes. "CHARACTERIZATION OF RECOMBINANT HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR MISSING THE FINGER DOMAIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643842.
Full textAbstract:
Site-specific mutagenesis was used to produce a mutant form of tissue-type plasminogen activator (t-PA) which was missing the first 44 amino acids. This domain has sequence homology with the type 1 regions in proteins such as fibronectin and is commonly called the finger domain. The mutant protein (des 1-44 t-PA) was expressed in Chinese Hampster Ovary cells, and was purified using chromatography on Zn-chelate sepharose and lysine-sepharose. Sequence analysis indicated that the resulting protein was homogeneous and started at amino acid 45 in the sequence of the normal protein. The two-chain forms of both des 1-44 t-PA and normal sequence t-PA exhibited similar kinetic constants with a small synthetic substrate (H-D-Isoleucyl-L- prolyl-L-arginyl-p-nitroani 1 ide). The ability of des 1-44 t-PA to activate plasminogen was decreased to 70% of the rate of normal t-PA. The rate of plasminogen activation by normal t-PA was stimulated 51-fold in the presence of fibrin, whereas with des 1-44 t-PA it was stimulated only 40-fold. Although des 1-44 t-PA bound to lysine-agarose, little (if any) binding was observed to either intact or degraded fibrin indicating that fibrin stimulation is due in part to the ability of t-PA to recognize plasminogen bound to fibrin as a preferable substrate. The mutant t-PA was capable of forming complexes in vitro with all of the inhibitors in blood which react with normal sequence t-PA. The rate of reaction with α2-macroglobulin, however, was slower with des 1-44 t-PA than with normal sequence t-PA. The similar resistance of des 1-44 t-PA and normal sequence t-PA to proteolysis and the ability to react with a battery of monoclonal antibodies suggests that the deletion did not cause perturbed folding, but rather that alterations in function of des 1-44 t-PA were due to the lack of the finger domain.
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de Vries, C. J. M., N. K. Veerman, and H. Pannekoek. "ARTIFICIAL EXON SHUFFLING: CONSTRUCTION OF HYBRID cDNAS CONTAINING DOMAINS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (T-PA) AND UROKINASE (u-PA)." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643940.
Full textAbstract:
The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling
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Jorgensen, M. J., MJ Rabiet, A. B. Cantor, B. Furie, C. L. Brown, C. B. Shoemaker, and B. C. Furie. "VITAMIN K-DEPENDENT γ-CARBOXYLATION OF FACTOR IX REQUIRES A RECOGNITION SITE CONTAINED WITHIN THE PROPEPTIDE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643564.
Full textAbstract:
The vitamin K-dependent proteins, including Factor IX (FIX), are calcium-binding proteins that undergo vitamin K-dependent post-translational modification to convert amino terminal glutamic aoid residues to Gla residues. Sequence homology among the propeptides of these proteins suggests a role for this region in designating the adjacent glutamic acid-rich domain for γ-carboxylation during intraoellular processing. Mutations vere made in the propeptide (residues -1 to -18) of FIX, and the effects on γ-carboxylation were assessed. The human FIX cDNA coding sequenoe was modified using oligonucleotide-directed site-specific mutagenesis and was expressed in Chinese hamster ovary cells. The extent of γ-carboxylation of secreted FIX was determined by (1) ability to interact with conformation-specific antibodies directed against the Gla-dependent, metal-stabilized, native structure of FIX, and (2) direct Gla analysis of the alkaline hydrolysate. Using the unmodified coding sequence, 64 ± 17 % of recombinant Factor IX bound to the conformation-specific antibodies, and 9.4 ± 0.7 Gla residues were found (compared with 12 Gla in plasma FIX). When the 18-residue propeptide was deleted, secreted FIX contained no detectable native FIX antigen and no detectable Gla. Similarly, point mutations leading to substitution of Ala for Phe at residue -16 or Glu for Ala at residue -10 led to secretion of FIX containing 2% and 6% native antigen, respectively, and approximately 1-2 Gla residues. The molecular weight of each of the reoombinant FIX species, as estimated by SDS-PAGE, was identical to that of plasma FIX. NH2-terminal sequence analysis of the mutant FIX speoies yielded the NH2-terminal sequence of plasma FIX. These data indicate that the mutations made in the propeptide did not interfere with intracellular proteolytic prooessing of FIX. We conolude that the FIX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for γ-carboxylation. The association of the propeptide with the γ-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in post-translational protein processing.
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Pannekok, H., A. J. Van Zonneveid, C. J. M. de vries, M. E. MacDonald, H. Veerman, and F. Blasi. "FUNCTIONAL PROPERTIES OF DELETION-MUTANTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643724.
Full textAbstract:
Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.
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Sadler, J. Evan. "THE MOLECULAR BIOLOGY OF VON WILLEBRAND FACTOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643930.
Full textAbstract:
Human von Willebrand factor (vWF) is a plasma glycoprotein that is synthesized by endothelial cells and megakaryocytes, and perhaps by syncytiotrophoblast of placenta. The biosynthesis of vWF is very complex, involving proteolytic processing, glycosyla-tion, disulfide bond formation, and sulfation. Mature vWF consists of a single subunit of ∼ 250,000 daltons that is assembled into multimer ranging from dimers to species of over 10 million daltons. vWF performs its essential hemostatic function through several binding interactions, forming a bridge between specific receptors on the platelet surface and components of damaged vascular subendothelial connective tissue. Inherited deficiency of vWF, or von Willebrand disease (vWD), is the most common genetically transmitted bleeding disorder worldwide. The last two years has been a time of very rapid progress in understanding the molecular biology of vWF. Four research groups have independently isolated and sequenced the 9 kilobase full-length vWF cDNA. The predicted protein sequence has provided a foundation for understanding the biosynthetic processing of vWF, and has clarified the relationship between vWF and a 75-100 kilodalton plasma protein of unknown function, von Willebrand antigen II (vWAgll)/ vWAgll is co-distributed with vWF in endothelial cells and platelets, and is deficient in patients with vWD. The cDNA sequence of vWF shows that vWAgll is a rather large pro-peptide for vWF, explaining the biochemical and genetic association between the two proteins. vWF has a complex evolutionary history marked by many separate gene segment duplications. The primary structure of the protein contains four distinct types of repeated domains present in two to four copies each. Repeated domains account for over 90 percent of the protein sequence. This sequence provides a framework for ordering the functional domains that have been defined by protein chemistry methods. A tryptic peptide from the amino-terminus of vWF that overlaps domain D3 binds to factor VIII and also appears to bind to heparin. Peptides that include domain A1 bind to collagens, to heparin, and to platelet glycoprotein Ib. A second collagen binding site appears to lie within domain A3. The vWF cDNA has been expressed in heterologous cells to produce small amounts of functionally and structurally normal vWF, indicating that endothelial cells are not unique in their ability to process and assemble vWF multimers. Site-directed mutagenesis has been used to show that deletion of the propeptide of vWF prevents the formation of multimers. Cloned cDNA probes have been employed to isolate vWF genomic DNA from cosmid and λ-phage libraries, and the size of the vWF gene appears to be ∼ 150 kilobases. The vWF locus has been localized to human chromosome 12p12—pter. Several intragenic RFLPs have been characterized. With them, vWF has been placed on the human genetic linkage map as the most telomeric marker currently available for the short arm of chromosome 12. A second apparently homologous locus has been identified on chromosome 22, but the relationship of this locus to the authentic vWF gene is not yet known. The mechanism of vWD has been studied by Southern blotting of genomic DNA with cDNA probes in a few patients. Three unrelated pedigrees have been shown to have total deletions of the vWF gene as the cause of severe vWD (type III). This form of gene deletion appears to predispose to the development of inhibitory alloantibodies to vWF during therapy with cryoprecipitate. During the next several years recombinant DNA methods will continue to contribute our understanding of the evolution, biosynthesis, and structure-function relationships of vWF, as well as the mechanism of additional variants of vWD at the level of gene structure.