103 results on '"Levisson, Mark"'
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2. Microbial production of short and medium chain esters: Enzymes, pathways, and applications
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Kruis, Aleksander J., Bohnenkamp, Anna C., Patinios, Constantinos, van Nuland, Youri M., Levisson, Mark, Mars, Astrid E., van den Berg, Corjan, Kengen, Servé W.M., and Weusthuis, Ruud A.
- Published
- 2019
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3. Monascus ruber as cell factory for lactic acid production at low pH
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Weusthuis, Ruud A., Mars, Astrid E., Springer, Jan, Wolbert, Emil JH, van der Wal, Hetty, de Vrije, Truus G., Levisson, Mark, Leprince, Audrey, Houweling-Tan, G.Bwee, PHA Moers, Antoine, Hendriks, Sjon NA, Mendes, Odette, Griekspoor, Yvonne, Werten, Marc WT, Schaap, Peter J., van der Oost, John, and Eggink, Gerrit
- Published
- 2017
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4. Ethyl acetate production by the elusive alcohol acetyltransferase from yeast
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Kruis, Aleksander J., Levisson, Mark, Mars, Astrid E., van der Ploeg, Max, Garcés Daza, Fernando, Ellena, Valeria, Kengen, Servé W.M., van der Oost, John, and Weusthuis, Ruud A.
- Published
- 2017
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5. Engineering de novo anthocyanin production in Saccharomyces cerevisiae
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Levisson, Mark, Patinios, Constantinos, Hein, Sascha, de Groot, Philip A., Daran, Jean-Marc, Hall, Robert D., Martens, Stefan, and Beekwilder, Jules
- Published
- 2018
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6. A metal ion charged mixed matrix membrane for selective adsorption of hemoglobin
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Tetala, Kishore K.R., Skrzypek, Katarzyna, Levisson, Mark, and Stamatialis, Dimitrios F.
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- 2013
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7. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site
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Sayer, Christopher, Finnigan, William, Isupov, Michail N., Levisson, Mark, Kengen, Servé W. M., van der Oost, John, Harmer, Nicholas J., and Littlechild, Jennifer A.
- Published
- 2016
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8. Carboxylic ester hydrolases from hyperthermophiles
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Levisson, Mark, van der Oost, John, and Kengen, Servé W. M.
- Published
- 2009
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9. Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis
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Wolterink-van Loo, Suzanne, Levisson, Mark, Cabrières, Maud C., Franssen, Maurice C. R., and van der Oost, John
- Published
- 2008
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10. Novel routes towards bioplastics from plants : elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes
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Jongedijk, Esmer, Muller, Sebastian, van Dijk, Aalt D. J., Schijlen, Elio, Champagne, Antoine, Boutry, Marc, Levisson, Mark, van der Krol, Sander, Bouwmeester, Harro, Beekwilder, Jules, Jongedijk, Esmer, Muller, Sebastian, van Dijk, Aalt D. J., Schijlen, Elio, Champagne, Antoine, Boutry, Marc, Levisson, Mark, van der Krol, Sander, Bouwmeester, Harro, and Beekwilder, Jules
- Abstract
Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (-)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.
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- 2020
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11. (Hyper)thermophilic enzymes : Production and purification
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Falcicchio, Pierpaolo, Levisson, Mark, Kengen, Servé W.M., Koutsopoulos, Sotirios, van der Oost, John, Falcicchio, Pierpaolo, Levisson, Mark, Kengen, Servé W.M., Koutsopoulos, Sotirios, and van der Oost, John
- Abstract
The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where “normal” proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.
- Published
- 2020
12. Biochemical and Structural Analysis of Thermostable Esterases
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Levisson, Mark, Servé Kengen, and Oost, John Van Der
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- 2019
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13. Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima
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Levisson, Mark, van der Oost, John, and Kengen, Servé W. M.
- Published
- 2007
14. Toward developing a yeast cell factory for the production of prenylated flavonoids
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Levisson, Mark (author), Araya-Cloutier, Carla (author), De Bruijn, Wouter J.C. (author), Van Der Heide, Menno (author), Salvador López, José Manuel (author), Daran, J.G. (author), Vincken, Jean Paul (author), Beekwilder, Jules (author), Levisson, Mark (author), Araya-Cloutier, Carla (author), De Bruijn, Wouter J.C. (author), Van Der Heide, Menno (author), Salvador López, José Manuel (author), Daran, J.G. (author), Vincken, Jean Paul (author), and Beekwilder, Jules (author)
- Abstract
Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae. As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L -1 (0.35 μM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed., BT/Industrial Microbiology
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- 2019
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15. Toward developing a yeast cell factory for the production of prenylated flavonoids
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Levisson, Mark, Araya-Cloutier, Carla, Bruijn, Wouter J.C., De, Heide, Menno, Van Der, Salvador López, José Manuel, Daran, Jean Marc, Vincken, Jean Paul, Beekwilder, Jules, Levisson, Mark, Araya-Cloutier, Carla, Bruijn, Wouter J.C., De, Heide, Menno, Van Der, Salvador López, José Manuel, Daran, Jean Marc, Vincken, Jean Paul, and Beekwilder, Jules
- Abstract
Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae. As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L -1 (0.35 μM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.
- Published
- 2019
16. Salvia dorisiana
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Jongedijk, Esmer, Müller, Sebastian, Van Dijk, Aalt D.J., Schijlen, Elio, Champagne, Antoine, Boutry, Marc, Levisson, Mark, Van Der Krol, Sander, Bouwmeester, Harro, Beekwilder, Jules, Jongedijk, Esmer, Müller, Sebastian, Van Dijk, Aalt D.J., Schijlen, Elio, Champagne, Antoine, Boutry, Marc, Levisson, Mark, Van Der Krol, Sander, Bouwmeester, Harro, and Beekwilder, Jules
- Abstract
Gene expression in different tissues of Salvia dorisiana, Gene expression in different tissues of Salvia dorisiana
- Published
- 2019
17. MOESM5 of Engineering de novo anthocyanin production in Saccharomyces cerevisiae
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Levisson, Mark, Constantinos Patinios, Hein, Sascha, Groot, Philip, Jean-Marc Daran, Hall, Robert, Martens, Stefan, and Beekwilder, Jules
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GeneralLiterature_INTRODUCTORYANDSURVEY - Abstract
Additional file 5. Table with primers used in this study. Table with primers used in this study.
- Published
- 2018
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18. Engineering de novo anthocyanin production in Saccharomyces cerevisiae
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Levisson, Mark (author), Patinios, Constantinos (author), Hein, Sascha (author), de Groot, P.A. (author), Daran, J.G. (author), Martens, Stefan (author), Hall, Robert D. (author), Beekwilder, Jules (author), Levisson, Mark (author), Patinios, Constantinos (author), Hein, Sascha (author), de Groot, P.A. (author), Daran, J.G. (author), Martens, Stefan (author), Hall, Robert D. (author), and Beekwilder, Jules (author)
- Abstract
Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. Results: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 μmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 μM [5 mg/L] and 150 μM [44 mg/L], respectively. Conclusion: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current, Applied Sciences, BT/Industrial Microbiology
- Published
- 2018
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19. RNAseq of Salvia dorisiana plant parts
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Jongedijk, Esmer, Müller, Sebastian, Van Dijk, Aalt D.J., Schijlen, Elio, Champagne, Antoine, Boutry, Marc, Levisson, Mark, Van Der Krol, Sander, Bouwmeester, Harro, Beekwilder, Jules, Jongedijk, Esmer, Müller, Sebastian, Van Dijk, Aalt D.J., Schijlen, Elio, Champagne, Antoine, Boutry, Marc, Levisson, Mark, Van Der Krol, Sander, Bouwmeester, Harro, and Beekwilder, Jules
- Abstract
RNAseq of Salvia dorisiana plant parts Overall design: RNAseq of Salvia dorisiana plant parts, RNAseq of Salvia dorisiana plant parts Overall design: RNAseq of Salvia dorisiana plant parts
- Published
- 2018
20. vanI : a novel D-Ala-D-Lac vancomycin resistance gene cluster found in Desulfitobacterium hafniense
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Kruse, Thomas, Levisson, Mark, de Vos, Willem M, Smidt, Hauke, Departments of Faculty of Veterinary Medicine, Veterinary Biosciences, Haartman Institute (-2014), Department of Bacteriology and Immunology, and de Vos & Salonen group
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DNA, Bacterial ,enrichment ,genome sequence ,Molecular Sequence Data ,education ,COMPARATIVE-ANALYSIS SYSTEM ,Desulfitobacterium ,413 Veterinary science ,Microbiology ,Microbiologie ,RESISTOME ,evolution ,Cluster Analysis ,STREPTOMYCES-COELICOLOR ,comparative-analysis system ,bacteria ,resistome ,Research Articles ,Phylogeny ,VLAG ,WIMEK ,ENTEROCOCCUS-FAECIUM ,GENOME SEQUENCE ,Vancomycin Resistance ,alignment ,ANTIBIOTIC-RESISTANCE ,Sequence Analysis, DNA ,streptomyces-coelicolor ,EVOLUTION ,ALIGNMENT ,antibiotic-resistance ,Multigene Family ,BACTERIA ,3111 Biomedicine ,enterococcus-faecium ,ENRICHMENT - Abstract
The glycopeptide vancomycin was until recently considered a drug of last resort against Gram-positive bacteria. Increasing numbers of bacteria, however, are found to carry genes that confer resistance to this antibiotic. So far, 10 different vancomycin resistance clusters have been described. A chromosomal vancomycin resistance gene cluster was previously described for the anaerobic Desulfitobacterium hafniense Y51. We demonstrate that this gene cluster, characterized by its d-Ala-d-Lac ligase-encoding vanI gene, is present in all strains of D. hafniense, D. chlororespirans and some strains of Desulfosporosinus spp. This gene cluster was not found in vancomycin-sensitive Desulfitobacterium or Desulfosporosinus spp., and we show that this antibiotic resistance can be exploited as an intrinsic selection marker for Desulfitobacterium hafniense and D. chlororespirans. The gene cluster containing vanI is phylogenetically only distantly related with those described from soil and gut bacteria, but clusters instead with vancomycin resistance genes found within the phylum Actinobacteria that include several vancomycin-producing bacteria. It lacks a vanH homologue, encoding a D-lactate dehydrogenase, previously thought to always be present within vancomycin resistance gene clusters. The location of vanH outside the resistance gene cluster likely hinders horizontal gene transfer. Hence, the vancomycin resistance cluster in D. hafniense should be regarded a novel one that we here designated vanI after its unique d-Ala-d-Lac ligase.
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- 2014
21. Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches
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Ruigrok, Vincent J. B., Levisson, Mark, Hekelaar, Johan, Smidt, Hauke, Dijkstra, Bauke W., van der Oost, John, Ruigrok, Vincent J. B., Levisson, Mark, Hekelaar, Johan, Smidt, Hauke, Dijkstra, Bauke W., and van der Oost, John
- Abstract
Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (K-D). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.
- Published
- 2012
22. (Hyper)thermophilic Enzymes: Production and Purification.
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Falcicchio, Pierpaolo, Levisson, Mark, Kengen, Servé W. M., and Koutsopoulos, Sotirios
- Abstract
The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where ˵normal″ proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in
E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications. [ABSTRACT FROM AUTHOR]- Published
- 2014
- Full Text
- View/download PDF
23. Phage Display of Engineered Binding Proteins.
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Levisson, Mark, Spruijt, Ruud B., Winkel, Ingrid Nolla, Kengen, Servé W. M., and van der Oost, John
- Abstract
In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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24. Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima.
- Author
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Levisson, Mark, Han, Gye Won, Deller, Marc C., Xu, Qingping, Biely, Peter, Hendriks, Sjon, Ten Eyck, Lynn F., Flensburg, Claus, Roversi, Pietro, Miller, Mitchell D., McMullan, Daniel, von Delft, Frank, Kreusch, Andreas, Deacon, Ashley M., van der Oost, John, Lesley, Scott A., Elsliger, Marc-André, Kengen, Servé W. M., and Wilson, Ian A.
- Abstract
TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β- D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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25. Kinetic and Stoichiometric Characterisation of Streptavidin-Binding Aptamers.
- Author
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Ruigrok, Vincent J. B., van Duijn, Esther, Barendregt, Arjan, Dyer, Kevin, Tainer, John A., Stoltenburg, Regina, Strehlitz, Beate, Levisson, Mark, Smidt, Hauke, and van der Oost, John
- Published
- 2012
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26. Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain
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Levisson, Mark, Sun, Lei, Hendriks, Sjon, Swinkels, Peter, Akveld, Twan, Bultema, Jelle B., Barendregt, Arjan, van den Heuvel, Robert H.H., Dijkstra, Bauke W., van der Oost, John, and Kengen, Servé W.M.
- Subjects
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CRYSTALS , *ESTERASES , *IMMUNOGLOBULINS , *BACTERIAL genetics , *ESCHERICHIA coli , *PROTEIN structure , *ELECTRON microscopy - Abstract
Summary: Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 °C and at pH 8.5, with a preference for esters with short acyl chains (C2–C10). Its 2.6-Å-resolution crystal structure revealed a classical α/β hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-Å-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of EstA. [Copyright &y& Elsevier]
- Published
- 2009
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27. Regulation of Pentose Catabolic Pathway Genes of Aspergillus niger.
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De Groot, Marco J. L., Van Den Dool, Carline, Wösten, Han A. B., Levisson, Mark, VanKuyk, Patricia A., Ruijter, George J. G., and De Vries, Ronald P.
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ASPERGILLUS niger ,PENTOSES ,METABOLISM ,GENES ,XYLITOL ,DEHYDROGENASES - Abstract
The aim of this study was to obtain a better understanding of the pentose catabolism in Aspergillus niger and the regulatory systems that affect it. To this end, we have cloned and characterised the genes encoding A. niger L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA), and compared the regulation of these genes to other genes of the pentose catabolic pathway. This demonstrated that activation of the pathway depends on two transcriptional regulators, the xylanolytic activator (XInR) and an unidentified C-arabinose specific regulator (AraR). These two regulators affect those genes of the pentose catabolic pathway that are related to catabolic conversion of their corresponding inducers (D-xylose and L-arabinose, respectively). [ABSTRACT FROM AUTHOR]
- Published
- 2007
28. Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima.
- Author
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Lei Sun, Levisson, Mark, Hendriks, Sjon, Akveld, Twan, Kengen, Servé W. M., Dijkstra, Bauke W., and van der Oost, John
- Subjects
- *
ESTERASES , *ESCHERICHIA coli , *POLYETHYLENE glycol , *CRYSTALLIZATION , *CRYSTALLOGRAPHY - Abstract
A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 Å resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 Å. There are two molecules in the asymmetric unit, with a VM of 2.9 Å3 Da−1 and 58% solvent content. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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29. [Untitled]
- Author
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Sayer, Christopher, Finnigan, William, Isupov, Michail N., Levisson, Mark, Kengen, Servé W. M., van der Oost, John, Harmer, Nicholas J., and Littlechild, Jennifer A.
- Subjects
Models, Molecular ,0301 basic medicine ,Pantetheine ,Coenzyme A ,Molecular Conformation ,Biology ,Ligands ,Microbiology ,Esterase ,Article ,Substrate Specificity ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme activator ,Thioesterase ,Microbiologie ,Catalytic Domain ,Enzyme Stability ,Hydrolase ,Life Science ,Enzyme Inhibitors ,VLAG ,Multidisciplinary ,Esterases ,Archaeoglobus fulgidus ,Active site ,Hydrogen-Ion Concentration ,Enzyme Activation ,030104 developmental biology ,chemistry ,Biochemistry ,Solvents ,biology.protein ,Thermodynamics ,Hydrophobic and Hydrophilic Interactions ,Protein Binding - Abstract
A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.
30. Engineering de novo anthocyanin production in <italic>Saccharomyces cerevisiae</italic>.
- Author
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Levisson, Mark, Patinios, Constantinos, Hein, Sascha, de Groot, Philip A., Daran, Jean-Marc, Hall, Robert D., Martens, Stefan, and Beekwilder, Jules
- Subjects
ANTHOCYANINS ,SACCHAROMYCES cerevisiae ,BIOSYNTHESIS ,NARINGENIN ,ARABIDOPSIS thaliana ,GENOMES - Abstract
Background: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast
Saccharomyces cerevisiae , production of anthocyanins has not yet been reported. Results:Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O -glucoside starting from glucose. Specific anthocyanin biosynthetic genes fromArabidopsis thaliana andGerbera hybrida were introduced in aS. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O -glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O -glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O -glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW , while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively. Conclusion: The results reported in this study demonstrate the proof-of-concept thatS. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O -glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries. [ABSTRACT FROM AUTHOR]- Published
- 2018
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31. Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052.
- Author
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Raedts, John, Siemerink, Marco A. J., Levisson, Mark, van der Oost, John, and Kengen, Servé W. M.
- Subjects
- *
BUTANEDIOL , *NADPH oxidase , *REDUCTASE inhibitors , *ACETOIN derivatives , *DEHYDROGENASES , *CLOSTRIDIUM beijerinckii , *ESCHERICHIA coli - Abstract
Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn2+ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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- View/download PDF
32. (Hyper)Thermophilic Enzymes: Production and Purification.
- Author
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Falcicchio P, Levisson M, Kengen SWM, Koutsopoulos S, and van der Oost J
- Subjects
- Enzyme Stability, Escherichia coli enzymology, Escherichia coli genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Enzymes chemistry, Enzymes genetics, Enzymes isolation & purification, Escherichia coli chemistry, Hot Temperature
- Abstract
The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.
- Published
- 2021
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- View/download PDF
33. Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes.
- Author
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Jongedijk E, Müller S, van Dijk ADJ, Schijlen E, Champagne A, Boutry M, Levisson M, van der Krol S, Bouwmeester H, and Beekwilder J
- Subjects
- Biosynthetic Pathways genetics, Terpenes metabolism, Nicotiana, Salvia genetics, Trichomes metabolism
- Abstract
Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (-)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2020
- Full Text
- View/download PDF
34. Toward Developing a Yeast Cell Factory for the Production of Prenylated Flavonoids.
- Author
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Levisson M, Araya-Cloutier C, de Bruijn WJC, van der Heide M, Salvador López JM, Daran JM, Vincken JP, and Beekwilder J
- Subjects
- Arylsulfotransferase genetics, Arylsulfotransferase metabolism, Metabolic Engineering, Plant Proteins genetics, Plant Proteins metabolism, Prenylation, Sophora enzymology, Flavonoids biosynthesis, Flavonoids chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae . As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L
-1 (0.35 μM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.- Published
- 2019
- Full Text
- View/download PDF
35. vanI: a novel D-Ala-D-Lac vancomycin resistance gene cluster found in Desulfitobacterium hafniense.
- Author
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Kruse T, Levisson M, de Vos WM, and Smidt H
- Subjects
- Cluster Analysis, DNA, Bacterial chemistry, DNA, Bacterial genetics, Molecular Sequence Data, Phylogeny, Sequence Analysis, DNA, Desulfitobacterium drug effects, Desulfitobacterium genetics, Multigene Family, Vancomycin Resistance
- Abstract
The glycopeptide vancomycin was until recently considered a drug of last resort against Gram-positive bacteria. Increasing numbers of bacteria, however, are found to carry genes that confer resistance to this antibiotic. So far, 10 different vancomycin resistance clusters have been described. A chromosomal vancomycin resistance gene cluster was previously described for the anaerobic Desulfitobacterium hafniense Y51. We demonstrate that this gene cluster, characterized by its d-Ala-d-Lac ligase-encoding vanI gene, is present in all strains of D. hafniense, D. chlororespirans and some strains of Desulfosporosinus spp. This gene cluster was not found in vancomycin-sensitive Desulfitobacterium or Desulfosporosinus spp., and we show that this antibiotic resistance can be exploited as an intrinsic selection marker for Desulfitobacterium hafniense and D. chlororespirans. The gene cluster containing vanI is phylogenetically only distantly related with those described from soil and gut bacteria, but clusters instead with vancomycin resistance genes found within the phylum Actinobacteria that include several vancomycin-producing bacteria. It lacks a vanH homologue, encoding a D-lactate dehydrogenase, previously thought to always be present within vancomycin resistance gene clusters. The location of vanH outside the resistance gene cluster likely hinders horizontal gene transfer. Hence, the vancomycin resistance cluster in D. hafniense should be regarded a novel one that we here designated vanI after its unique d-Ala-d-Lac ligase., (© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)
- Published
- 2014
- Full Text
- View/download PDF
36. Phage display of engineered binding proteins.
- Author
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Levisson M, Spruijt RB, Winkel IN, Kengen SW, and van der Oost J
- Subjects
- Recombinant Proteins genetics, Bacteriophage M13 genetics
- Abstract
In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.
- Published
- 2014
- Full Text
- View/download PDF
37. (Hyper)thermophilic enzymes: production and purification.
- Author
-
Falcicchio P, Levisson M, Kengen SW, and Koutsopoulos S
- Subjects
- Biocatalysis, Chromatography, Affinity, Chromatography, Gel, Enzymes metabolism, Enzymes biosynthesis, Enzymes isolation & purification
- Abstract
The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how is structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.
- Published
- 2014
- Full Text
- View/download PDF
38. Characterization of aptamer-protein complexes by X-ray crystallography and alternative approaches.
- Author
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Ruigrok VJB, Levisson M, Hekelaar J, Smidt H, Dijkstra BW, and Van der Oost J
- Subjects
- Animals, Crystallography, X-Ray, Humans, Models, Molecular, Nucleic Acid Conformation, Protein Conformation, Aptamers, Nucleotide chemistry, Aptamers, Nucleotide metabolism, Proteins chemistry, Proteins metabolism
- Abstract
Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (K(D)). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.
- Published
- 2012
- Full Text
- View/download PDF
39. Alternative affinity tools: more attractive than antibodies?
- Author
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Ruigrok VJ, Levisson M, Eppink MH, Smidt H, and van der Oost J
- Subjects
- Animals, Chromatography, Affinity, Drug Delivery Systems methods, Drug Delivery Systems trends, Humans, Nucleic Acids chemistry, Protein Engineering, Antibodies chemistry, Aptamers, Nucleotide chemistry, Carrier Proteins chemistry, Molecular Imprinting
- Abstract
Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.
- Published
- 2011
- Full Text
- View/download PDF
40. Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima.
- Author
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Sun L, Levisson M, Hendriks S, Akveld T, Kengen SW, Dijkstra BW, and van der Oost J
- Subjects
- Bacterial Proteins isolation & purification, Binding Sites, Cloning, Molecular, Crystallization, Crystallography, X-Ray, DNA, Bacterial chemistry, DNA, Bacterial genetics, Escherichia coli genetics, Esterases isolation & purification, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Bacterial Proteins chemistry, Esterases chemistry, Thermotoga maritima enzymology
- Abstract
A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 A resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 A. There are two molecules in the asymmetric unit, with a V(M) of 2.9 A3 Da(-1) and 58% solvent content.
- Published
- 2007
- Full Text
- View/download PDF
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