Biomedical subjects

Mathias Uhlén

Publications and source records attributed to Mathias Uhlén.

At least 19 recordsLinked to original sources

PubMed2005

Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana.

BACKGROUND: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. RESULTS: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes. CONCLUSIONS: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21,000 Arabidopsis transcripts.

Arabidopsis↗

PubMed2005

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures.

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70 degrees C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology.

Autoanalysis↗

PubMed2004

A Populus EST resource for plant functional genomics.

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.

Ecosystem↗

PubMed2004

Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts.

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Antibodies↗

PubMed2004

Genome-based proteomics.

Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.

Chromatography, Liquid↗

PubMed2004

Transcriptional responses of Paxillus involutus and Betula pendula during formation of ectomycorrhizal root tissue.

In order to obtain information on genes specifically expressed in the ectomycorrhizal symbiosis, 3,555 expressed sequence tags (ESTs) were analyzed from a cDNA library constructed from ectomycorrhiza formed between the basidiomycete Paxillus involutus and birch (Betula pendula). cDNA libraries from saprophytically growing fungus (3,964 ESTs) and from axenic plants (2,532 ESTs) were analyzed in parallel. By clustering all the EST obtained, a nonredundant set of 2,284 unique transcripts of either fungal or plant origin were identified. The expression pattern of these genes was analyzed using cDNA microarrays. The analyses showed that the plant and fungus responded to the symbiosis by altering the expression levels of a number of enzymes involved in carbon metabolism. Several plant transcripts with sequence similarities to genes encoding enzymes in the tricarboxylic cycle and electron transport chain were down regulated as compared with the levels in free-living roots. In the fungal partner, a number of genes encoding enzymes in the lipid and secondary metabolism were down regulated in mycorrhiza as compared with the saprophytically growing mycelium. A substantial number of the ESTs analyzed displayed significant sequence similarities to proteins involved in biotic stress responses, but only a few of them showed differential expression in the mycorrhizal tissue, including plant and fungal metallothioneins and a plant defensin homologue. Several of the genes that were differentially expressed in the mycorrhizal root tissue displayed sequence similarity to genes that are known to regulate growth and development of plant roots and fungal hyphae, including transcription factors and Rho-like GTPases.

Amino Acid Sequence↗

PubMed2004

The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP).

Mycoplasma mycoides subsp. mycoidesSC (MmymySC)is the etiological agent of contagious bovine pleuropneumonia (CBPP), a highly contagious respiratory disease in cattle. The genome of Mmymy SC type strain PG1(T) has been sequenced to map all the genes and to facilitate further studies regarding the cell function of the organism and CBPP. The genome is characterized by a single circular chromosome of 1211703 bp with the lowest G+C content (24 mole%)and the highest density of insertion sequences (13% of the genome size)of all sequenced bacterial genomes. The genome contains 985 putative genes, of which 72 are part of insertion sequences and encode transposases. Anomalies in the GC-skew pattern and the presence of large repetitive sequences indicate a high genomic plasticity. A variety of potential virulence factors was identified, including genes encoding putative variable surface proteins and enzymes and transport proteins responsible for the production of hydrogen peroxide and the capsule, which is believed to have toxic effects on the animal.

Animals↗

PubMed2004

Rapid combined characterization of microorganism and host genotypes using a single technology.

BACKGROUND: Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrosequencing trade mark technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection. MATERIALS AND METHODS: DNA from 87 clinical isolates of H. pylori, 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples. RESULTS: All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called. CONCLUSIONS: We conclude that genetic analysis using Pyrosequencing trade mark technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markers.

Animals↗

PubMed2003

Microbead display of proteins by cell-free expression of anchored DNA.

Gene expression technologies where nucleic acid sequences remain physically linked to their corresponding gene products are important tools for selection and identification of rare variants in large protein libraries. Here, we describe a gene expression system, which combines the potential of bead-based suspension array technology (SAT) with gene expression and clonal identification. Using streptavidin-coated polystyrene micrometer-sized beads as solid supports for anchored PCR products, we have investigated conditions for cell-free expression and bioaffinity technology to provide clonal co-anchoring of corresponding gene products. Experiments showed that coupled transcription and translation of PCR product expression cassettes resulted in display of affinity-anchored proteins whose binding characteristics could be analyzed via direct and selective interaction with a fluorescently labeled target protein. Interestingly, experiments performed with differently biotinylated PCR products showed that the efficiency of display was dependent on the directionality of the expression cassette relative to the bead surface. In spiked systems, using small immunoglobulin binding proteins as models, we demonstrate efficient flow cytometric sorting of beads corresponding to the target interacting clones, verified by post-sorting analysis and clonal identification at DNA level. The use of this technology, including alternative formats, for different proteomics applications is discussed.

Adsorption↗

PubMed2003

Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays.

Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.

DNA Primers↗

PubMed2003

Affibody-beta-galactosidase immunoconjugates produced as soluble fusion proteins in the Escherichia coli cytosol.

Recombinant immunoconjugates constitute a novel class of immunoassay reagents produced by genetic fusion between an antigen recognizing moiety and a reporter enzyme or fluorescent protein, obviating the need for chemical coupling. In this work, we describe the construction, Escherichia coli production and characterization of recombinant beta-galactosidase (beta-gal)-based immunoconjugates directed to human immunoglobulin A (IgA). As the antigen recognizing moieties, either monovalent or dimeric (head-to-tail) versions of an IgA-specific affibody (Z(IgA1)) were used, previously selected in vitro from a protein library based on combinatorial engineering of a single staphylococcal protein A domain. To increase the likelihood of proper presentation on the assembled homotetrameric enzyme surface, the affibody moieties were linked to the N-terminus of the enzyme subunits via a heptapeptide linker sequence. The two resulting immunoconjugates Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal, containing four and eight affibody moieties per enzyme, respectively, could be expressed as soluble and proteolytically stable proteins intracellularly in E. coli from where they were purified to high purity by a single anion exchange chromatography step. The yields of immunoconjugates were in the range 200-400 mg/l culture. Biosensor-binding studies showed that both the Z(IgA1)-beta-gal and (Z(IgA1))(2)-beta-gal immunoconjugates were capable of selective IgA-recognition, but with an apparent higher binding affinity for the variant containing divalent affibody moieties, presumably due to avidity effects. The applicability of this class of recombinant immunoconjugates was demonstrated by IgA detection in enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. In addition, using human kidney biopsy samples from a nephropathy patient, IgA depositions in glomeruli could be detected by immunohistochemistry with low background staining of tissue.

Biosensing Techniques↗

PubMed2003

Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues.

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.

Antibodies↗

PubMed2002

A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products.

An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag Z(SPA-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 Z(SPA-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 (P(T7)) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His(6)) tag in frame with the Z(SPA-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.

Amino Acid Sequence↗

PubMed2002

Single-vector three-frame expression systems for affinity-tagged proteins.

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.

Bacterial Proteins↗

PubMed2002

Anti-idiotypic protein domains selected from protein A-based affibody libraries.

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.

Antibodies, Anti-Idiotypic↗

PubMed2002

Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology.

The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.

Adsorption↗

PubMed2002

Gene expression analysis by signature pyrosequencing.

We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.

Base Sequence↗

PubMed2002

ISMmy1, a novel insertion sequence of Mycoplasma mycoides subsp. mycoides small colony type.

A new insertion sequence, ISMmy1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC). The occurrence of ISMmy1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG1(T), the vaccine strain T1Sr49, and the strain Afadé, which all had unique patterns. ISMmy1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta(T) while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy1. This observation suggests horizontal transfer between MmymySC and M. bovis.

Amino Acid Sequence↗