| 1. |
- Agaton, C., et al.
(author)
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Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues
- 2003
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In: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 2, s. 405-
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Journal article (peer-reviewed)abstract
- Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.
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| 2. |
- Alterman, Mathias, et al.
(author)
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P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays
- 2001
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In: European Journal of Pharmaceutical Sciences. - : Elsevier. - 0928-0987 .- 1879-0720. ; 13:2, s. 203-212
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Journal article (peer-reviewed)abstract
- The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1′ benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 μM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1–100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.
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| 3. |
- Andersson, Anders, et al.
(author)
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A transcriptional timetable of autumn senescence
- 2004
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In: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 5:4, s. R24-
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Journal article (peer-reviewed)abstract
- Background We have developed genomic tools to allow the genus Populus (aspens and cottonwoods) to be exploited as a full-featured model for investigating fundamental aspects of tree biology. We have undertaken large-scale expressed sequence tag (EST) sequencing programs and created Populus microarrays with significant gene coverage. One of the important aspects of plant biology that cannot be studied in annual plants is the gene activity involved in the induction of autumn leaf senescence. Results On the basis of 36,354 Populus ESTs, obtained from seven cDNA libraries, we have created a DNA microarray consisting of 13,490 clones, spotted in duplicate. Of these clones, 12,376 (92%) were confirmed by resequencing and all sequences were annotated and functionally classified. Here we have used the microarray to study transcript abundance in leaves of a free-growing aspen tree (Populus tremula) in northern Sweden during natural autumn senescence. Of the 13,490 spotted clones, 3,792 represented genes with significant expression in all leaf samples from the seven studied dates. Conclusions We observed a major shift in gene expression, coinciding with massive chlorophyll degradation, that reflected a shift from photosynthetic competence to energy generation by mitochondrial respiration, oxidation of fatty acids and nutrient mobilization. Autumn senescence had much in common with senescence in annual plants; for example many proteases were induced. We also found evidence for increased transcriptional activity before the appearance of visible signs of senescence, presumably preparing the leaf for degradation of its components.
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| 4. |
- Graslund, S., et al.
(author)
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Single-vector three-frame expression systems for affinity-tagged proteins
- 2002
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In: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 215:1, s. 139-147
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Journal article (peer-reviewed)abstract
- An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.
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| 5. |
- Gräslund, Susanne, et al.
(author)
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A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
- 2002
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In: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 99:1, s. 41-50
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Journal article (peer-reviewed)abstract
- An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.
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| 6. |
- Larsson, M., et al.
(author)
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Expression profile viewer (ExProView) : A software tool for transcriptome analysis
- 2000
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In: Genomics. - : Elsevier BV. - 0888-7543 .- 1089-8646. ; 63:3, s. 341-353
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Journal article (peer-reviewed)abstract
- A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a virtual chip, in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.
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| 7. |
- Larsson, M., et al.
(author)
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High-throughput protein expression of cDNA products as a tool in functional genomics
- 2000
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In: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 143-157
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Journal article (peer-reviewed)abstract
- A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.
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| 8. |
- Persson, Henrik, et al.
(author)
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Utvärdering och utveckling av säkerhetssystem mot tillbakabrand i pellets- och flisutrustningar
- 2004
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Reports (peer-reviewed)abstract
- There is always some risk of incident associated with the combustion of solid fuels in automatically stoked boilers or stoves. These incidents are of essentially three types: smoke from the burner back into the boiler room (or back into the living room from pellet stoves), gas explosions in the combustion chamber and back burning in the stoker. There can be a number of reasons for these problems, and in many cases such incidents have not been investigated. The Swedish Building Regulations stipulate that firing equipment shall have adequate safety against fire. They also recommend that stokers/burners should have at least two independent safety systems against back burning. The objective of this project has been to analyse such incidents and to test safety systems for pellet and wood chip burners, in order better to understand how they work. It has also included evaluation of the“fireguard” water sprinkler system. The results from the tests with pellet burners showed that the burners that were tested provided relatively good protection against back burning. However, it was found that it was very important how and where the systems (temperature limiter, water sprinkler etc.) were fitted on the burners in order to ensure correct and effective operation of the safety system. One of the key question was also where to position electronic equipment on the burners in order to ensure that it is not exposed to temperatures higher than those for which it is designed. Over the years, there have been major problems with back burning in older designs of wood chip burners using old technology. The chip burner used in this project was of modern type. The test with simulated back burning showed that the water sprinkler stopped the fire. However, for greater safety the water sprinklers should have included two separate water container. The test with the pellets stove showed that the temperature sensor was of crucial importance for safety against back burning in the stove. Back burn occurred in one of the tests, but only after all the stove’s safety systems had been rendered inoperable. The “Fireguard” water sprinkler system performed well in our tests. However, the position of the temperature sensors, and the integrity of their thermal contact, was of crucial importance for how quickly the sprinklers responded. Another water sprinkler (Syr) showed itself to be very slow to respond when its sensor were in poor thermal contact with the surface of the fuel feed chute. Experience from the tests in this project has shown that it is relatively difficult to simulate back burning in boilers and pellet stoves, despite setting up very favourable conditions for such back burning. Although modern burners and pellet stoves are equipped with all the safety systems that can be expected, several incidents still happen every year. This means that continued analysis of incidents and development of safety systems is essential. The results from this project will be included when SP’s P-marking rules for pellet burners, pellet stoves and chip wood burners are next reviewed
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| 9. |
- Ronnmark, J., et al.
(author)
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Construction and characterization of affibody-Fc chimeras produced in Escherichia coli
- 2002
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In: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 261:02-jan, s. 199-211
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Journal article (peer-reviewed)abstract
- Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human I-G. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture, Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of artificial antibodies was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such artificial antibodies should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.
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| 10. |
- Sterky, Fredrik, et al.
(author)
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A Populus EST resource for plant functional genomics
- 2004
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:38, s. 13951-13956
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Journal article (peer-reviewed)abstract
- Trees present a life form of paramount importance for terrestrial ecosystems and human societies because of their ecological structure and physiological function and provision of energy and industrial materials. The genus Populus is the internationally accepted model for molecular tree biology. We have analyzed 102,019 Populus ESTs that clustered into 11,885 clusters and 12,759 singletons. We also provide >4,000 assembled full clone sequences to serve as a basis for the upcoming annotation of the Populus genome sequence. A public web-based EST database (POPULUSDB) provides digital expression profiles for 18 tissues that comprise the majority of differentiated organs. The coding content of Populus and Arabidopsis genomes shows very high similarity, indicating that differences between these annual and perennial angiosperm life forms result primarily from differences in gene regulation. The high similarity between Populus and Arabidopsis will allow studies of Populus to directly benefit from the detailed functional genomic information generated for Arabidopsis, enabling detailed insights into tree development and adaptation. These data will also valuable for functional genomic efforts in Arabidopsis.
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