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Arjen Schots

Publications and source records attributed to Arjen Schots.

12 recordsLinked to original sources
PubMed2006

Fluorescent T7 display phages obtained by translational frameshift.

Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3' end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.

Bacterial Proteins↗
PubMed2006

Plant expression of chicken secretory antibodies derived from combinatorial libraries.

Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes.

Animals↗
PubMed2006

A symbiont-independent endo-1,4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita.

Substituted xylan polymers constitute a major part of the hemicellulose fraction of plant cell walls, especially in monocotyledons. Endo-1,4-beta-xylanases (EC 3.2.1.8) are capable of hydrolyzing substituted xylan polymers into fragments of random size. Many herbivorous animals have evolved intimate relationships with endosymbionts to exploit their enzyme complexes for the degradation of xylan. Here, we report the first finding of a functional endo-1,4-beta-xylanase gene from an animal. The gene (Mi-xyl1) was found in the obligate plant-parasitic root-knot nematode Meloidogyne incognita, and encodes a protein that is classified as a member of glycosyl hydrolase family 5. The expression of Mi-xyl1 is localized in the subventral esophageal gland cells of the nematode. Previous studies have shown that M. incognita has the ability to degrade cellulose and pectic polysaccharides in plant cell walls independent of endosymbionts. Including our current data on Mi-xyl1, we show that the endogenous enzyme complex in root-knot nematode secretions targets essentially all major cell wall carbohydrates to facilitate a stealthy intercellular migration in the host plant.

Amino Acid Sequence↗
PubMed2005

Display and selection of chicken IgA Fab fragments.

Passive immune therapy is regaining interest to prevent and cure infectious diseases both in human and veterinary medicine. Therefore, systems are required that enable efficient targeted selection of antibodies originating from virtually any animal species. Here, a system for the selection of chicken IgA, using phage display, is described. A novel phagemid vector (pChick3) for the display and selection of chicken IgA antibodies in Fab format was developed. The functionality of pChick3 was demonstrated by construction of an immune antibody library using B cells from chickens infected with Eimeria acervulina. From this library, 10 different IgA fragments with specific binding to the E. acervulina antigen mix, the sporozoite or oocyst fractions were selected. These results demonstrate the efficiency and versatility of the pChick3 vector system that can readily be applied to construct libraries and subsequently select antibodies of the alpha isotype against a wide variety of pathogens and parasites.

Amino Acid Sequence↗
PubMed2005

Phage display-selected single-chain antibodies confer high levels of resistance against Tomato spotted wilt virus.

Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the field.

Amino Acid Sequence↗
PubMed2004

A functional polymeric immunoglobulin receptor in chicken (Gallus gallus) indicates ancient role of secretory IgA in mucosal immunity.

Animals are continuously threatened by pathogens entering the body through natural openings. Here we show that in chicken ( Gallus gallus ), secretory IgA (sIgA) protects the epithelia lining these natural cavities. A gene encoding a chicken polymeric Ig receptor ( GG-pIgR ), a key component of sIgA, was identified, and shown to be expressed in the liver, intestine and bursa of Fabricius. All motifs involved in pIgR function are present, with a highly conserved Ig-binding motif in the first Ig-like domain. Physical association of GG-pIgR with pIgA in bile and intestine demonstrates that this protein is a functional receptor. Thus, as shown for mammals, this receptor interacts with J-chain-containing polymeric IgA (pIgA) at the basolateral epithelial cell surface resulting in transcytosis and subsequent cleavage of the pIgR, releasing sIgA in the mucosal lumen. Interestingly, the extracellular portion of GG-pIgR protein comprises only four Ig-like domains, in contrast with the five domain structure found in mammalian pIgR genes. The second Ig-like domain of mammalian pIgR does not have an orthologous domain in the chicken gene. The presence of pIgR in chicken suggests that this gene has evolved before the divergence of birds and reptiles, indicating that secretory Igs may have a prominent role in first line defence in various non-mammalian species.

Amino Acid Sequence↗
PubMed2004

Plant degradation: a nematode expansin acting on plants.

Expansin proteins, which have so far been identified only in plants, rapidly induce extension of plant cell walls by weakening the non-covalent interactions that help to maintain their integrity. Here we show that an animal, the plant-parasitic roundworm Globodera rostochiensis, can also produce a functional expansin, which it uses to loosen cell walls when invading its host plant. As this nematode is known to be able to disrupt covalent bonds in plant cell walls, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.

Animals↗
PubMed2004

Towards sorting of biolibraries using single-molecule fluorescence detection techniques.

The selection of specific binding molecules like peptides and proteins from biolibraries using, for instance, phage display methods can be quite time-consuming. It is therefore desirable to develop a strategy that is much faster in selection and sorting of potential binders out of a biolibrary. In this contribution we separately discuss the current achievements in generation of biolibraries, single-molecule detection techniques and microfluidic devices. A high-throughput microfluidic platform is then proposed that combines the propulsion of liquid containing fluorescent components of the biolibrary through microchannels, single-molecule fluorescence photon burst detection and real-time sorting of positive hits.

Fluorescence↗
PubMed2002

Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy.

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.

Algorithms↗
PubMed2002

Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol.

Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fragment consists of the variable light and heavy chain domains from a mouse IgG antibody, which are connected by a flexible linker peptide. In each domain one disulfide bridge is present. The functionality of antibodies, which are normally secreted via the oxidizing environment of the endoplasmic reticulum, depends on the formation of intramolecular disulfide bridges. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. In contrast, transient expression in tobacco protoplasts resulted in a cytosolic scFv lacking disulfide bridges, which had a substantially reduced affinity for the antigen. This indicates that functionality rather than stability is determined by the presence of disulfide bridges in the in planta-expressed scFv antibody. The controversial observation of disulfide bond formation in the cytosol is discussed.

Amino Acid Sequence↗
PubMed2000

Nematode Parasitism Genes.

The ability of nematodes to live on plant hosts involves multiple parasitism genes. The most pronounced morphological adaptations of nematodes for plant parasitism include a hollow, protrusible stylet (feeding spear) connected to three enlarged esophageal gland cells that express products that are secreted into plant tissues through the stylet. Reverse genetic and expressed sequence tag (EST) approaches are being used to discover the parasitism genes expressed in nematode esophageal gland cells. Some genes cloned from root-knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes have homologues reported in genomic analyses of Caenorhabditis elegans and animal-parasitic nematodes. To date, however, the candidate parasitism genes endogenous to the esophageal glands of plant nematodes (such as the ß-1,4-endoglucanases) have their greatest similarity to microbial genes, prompting speculation that genes for plant parasitism by nematodes may have been acquired by horizontal gene transfer.

functional genomics↗