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Hans Söderlund

Publications and source records attributed to Hans Söderlund.

At least 19 recordsLinked to original sources
PubMed2006

Heterologous expression and site-directed mutagenesis studies of two Trichoderma harzianum chitinases, Chit33 and Chit42, in Escherichia coli.

Heterologous expression of two fungal chitinases, Chit33 and Chit42, from Trichoderma harzianum was tested in the different compartments and on the surface of Escherichia coli cells. Our goal was to find a fast and efficient expression system for protein engineering and directed evolution studies of the two fungal enzymes. Cytoplasmic overexpression resulted in both cases in inclusion body formation, where active enzyme could be recovered after refolding. Periplasmic expression of Chit33, and especially of Chit42, proved to be better suited for mutagenesis purposes. Recombinant chitinases from the periplasmic expression system showed activity profiles similar to those of the native proteins. Both chitinases also degraded a RET (resonance energy transfer) based bifunctionalized chitinpentaose substrate in a similar manner as reported for some putative exochitinases in the glycosyl hydrolase family 18, offering a sensitive way to assay their activities. We further demonstrated that Chit42 can also be displayed on E. coli surface and the enzymatic activity can be measured directly from the whole cells using methylumbelliferyl-chitinbioside as a substrate. The periplasmic expression and the surface display of Chit42, both offer a suitable expression system for protein engineering and activity screening in a microtiter plate scale. As a first mutagenesis approach we verified the essential role of the two carboxylic acid residues E172 (putative proton donor) and D170 (putative stabilizer) in the catalytic mechanism of Chit42, and additionally the role of the carboxylic acid E145 (putative proton donor) in the catalytic mechanism of Chit33.

Chitinases↗
PubMed2006

A highly sensitive and multiplexed method for focused transcript analysis.

We describe a novel, multiplexed method for focused transcript analysis of tens to hundreds of genes. In this method TRAC (transcript analysis with aid of affinity capture) mRNA targets, a set of amplifiable detection probes of distinct sizes and biotinylated oligo(dT) capture probe are hybridized in solution. The formed sandwich hybrids are collected on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted, optionally amplified by a PCR using a universal primer pair and detected with laser-induced fluorescence and capillary electrophoresis. The probes were designed by using a computer program developed for the purpose. The TRAC method was adapted to 96-well format by utilizing an automated magnetic particle processor. Here we demonstrate a simultaneous analysis of 18 Saccharomyces cerevisiae transcripts from two experimental conditions and show a comparison with a qPCR system. The sensitivity of the method is significantly increased by the PCR amplification of the hybridized and eluted probes. Our data demonstrate a bias-free use of at least 16 cycles of PCR amplification to increase probe signal, allowing transcript analysis from 2.5 ng of the total mRNA sample. The method is fast and simple and avoids cDNA conversion. These qualifications make it a potential, new means for routine analysis and a complementing method for microarrays and high density chips.

Amino Acid Sequence↗
PubMed2006

Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria.

The differences in faecal bacterial population between irritable bowel syndrome (IBS) and control subjects have been reported in several studies. The aim of the present study was to compare the predominant and clostridial faecal microbiota of IBS subjects and healthy controls by applying denaturing gradient gel electrophoresis (DGGE) and a recently developed multiplexed and quantitative hybridization-based technique, transcript analysis with the aid of affinity capture (TRAC). According to the results, the studied clostridial groups (Clostridium histolyticum, Clostridium coccoides-Eubacterium rectale, Clostridium lituseburense and Clostridium leptum) represented the dominant faecal microbiota of most of the studied subjects, comprising altogether 29-87% of the total bacteria as determined by the hybridized 16S rRNA. The C. coccoides-E. rectale group was the dominant subgroup of clostridia, contributing a mean of 43% of the total bacteria in control subjects and 30% (constipation type) to 50% (diarrhoea type) in different IBS symptom category subjects. The proportion of the C. coccoides-E. rectale group was found to be significantly lower in the constipation-type IBS subjects than in the control subjects. DNA-based PCR-DGGE and RNA-based RT-PCR-DGGE analyses targeted to the predominant bacterial population showed considerable biodiversity as well as uniqueness of the microbiota in each subject, in both control and IBS subject groups. The RT-PCR-DGGE profiles of the IBS subjects further indicated higher instability of the bacterial population compared to the control subjects. The observations suggest that clostridial microbiota, in addition to the instability of the active predominant faecal bacterial population, may be involved in IBS.

Adult↗
PubMed2006

PCR DGGE and RT-PCR DGGE show diversity and short-term temporal stability in the Clostridium coccoides-Eubacterium rectale group in the human intestinal microbiota.

As the Clostridium coccoides-Eubacterium rectale (Erec; clostridial phylogenetic cluster XIVa) group is one of the major groups of the human intestinal microbiota, DNA- and RNA-based population analysis techniques (denaturing gradient gel electrophoresis; DGGE) were developed and applied to assess the diversity and temporal stability (6 months-2 years) of this faecal clostridial microbiota in 12 healthy adults. The stability of the Erec group was compared with the stability of the predominant bacterial microbiota, which was also assessed with PCR-DGGE. In addition, the Erec group was quantified with a hybridization-based method. According to our results, the Erec group was diverse in each subject, but interindividual uniqueness was not as clear as that of the predominant bacteria. The Erec group was found to be temporally as stable as the predominant bacteria. Over 200 clones obtained from two samples proved the developed method to be specific. However, the amount of bacteria belonging to the Erec group was not related to the diversity of that same bacterial group. In conclusion, the newly developed DGGE method proved to be a valuable and specific tool for the direct assessment of the stability of the Erec group, demonstrating diversity in addition to short-term stability in most of the subjects studied.

Adult↗
PubMed2005

Rapid and multiplexed transcript analysis of microbial cultures using capillary electophoresis-detectable oligonucleotide probe pools.

A rapid assay for multiplex transcript analysis based on solution hybridization with pools of oligonucleotide probes was developed. In this assay called TRAC (transcript analysis with aid of affinity capture) the mRNAs to be studied are hybridized with gene-specific detection probe pools and biotinylated oligo(dT) and captured on streptavidin-coated magnetic particles. Unbound sample material and nonspecifically bound detection probes are removed and the target-specific probes are eluted and detected by capillary electrophoresis. Simultaneous treatment of 96 samples was automated using a magnetic bead particle processor. The assay enabled detection of in vitro transcribed RNA at the level of 30 amol (20 pg) and over a 300-fold linear range. Besides extracted RNA, crude cell lysates were directly used as samples. The assay was used for transcriptional analysis of selected mRNAs in the filamentous fungus Trichoderma reesei in two experimental conditions. TRAC analysis was highly reproducible, providing expression results that were consistent with conventional Northern blot analysis. The whole procedure starting from sample collecting can be carried out in 2 h, making this assay suitable for high-throughput analysis of a limited set of mRNAs e.g. in gene expression monitoring of production organism in microbial bioprocesses.

Culture Media↗
PubMed2005

Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture.

Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin-nucleic acid-probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA-specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 10(2). The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

Bacteria↗
PubMed2005

Development of a high-throughput format for solid-phase extraction of enantiomers using an immunosorbent in 384-well plates.

An antibody-based solid-phase extraction method for filtered 384-well plates was developed for a medical drug candidate having two enantiomeric forms in order to demonstrate the potential of the use of recombinant antibody fragments as specific and efficient immunosorbents. An immobilization method using a six-histidine tag of the antibody fragment and mild oxidation was applied in order to immobilize antibody fragments in an oriented and kinetically stable way that ensured high capacity of the antibody support. Phosphate buffer or plasma spiked with enantiomers were used as samples. Selective solid-phase extraction was followed by liquid chromatography-mass spectrometry analysis. Average recoveries for buffer and plasma samples ranged from 79 to 122% and 80 to 108%, respectively. Good linearity was observed in the concentration range of 30-3000 ng/mL of the enantiomer.

Antibodies↗
PubMed2005

One-step homogeneous immunoassay for small analytes.

We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naive scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes.

Amino Acid Sequence↗
PubMed2004

Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum.

We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (alpha/beta)(8)-barrel fold enzymes to recognize beta-1,4-galactosylated and alpha-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data. Our kinetic and inhibition studies are indicative of the substrate-assisted catalysis mechanism for both chitinases. Both T. harzianum chitinases are able to catalyze some transglycosylation reactions and cleave both simple chito-oligosaccharides and synthetically modified, beta-1,4-galactosylated and alpha-1,3-fucosylated chito-oligosaccharides. The cleavage data give experimental evidence that the two chitinases have differences in their substrate-binding sites, Chit42 apparently having a deeper substrate binding groove, which provides more tight binding of the substrate at subsites (-2-1-+1+2). On the other hand, some flexibility for the sugar recognition at subsites more distal from the cleavage point is allowed in both chitinases. A galactose unit can be accepted at the putative subsites -4 and -3 of Chit42, and at the subsite -4 of Chit33. Fucose units can be accepted as a branch at the putative -3 and -4 sites of Chit33 and as a branch point at -3 of Chit42. These data provide a good starting point for future protein engineering work aiming at chitinases with altered substrate-binding specificity.

Animals↗
PubMed2003

A functional genomics approach toward the understanding of secondary metabolism in plant cells.

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.

Acetates↗
PubMed2003

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae.

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.

Carrier Proteins↗
PubMed2003

Functional inactivation of the conserved Sem1p in yeast by intrabodies.

Intrabody technology was applied to characterize the function and intracellular localization of a highly conserved Saccharomyces cerevisiae Sem1 protein. DSS1, the mammalian homologue of Sem1p, is functionally conserved between yeast and mammalian cells, and in mammalian cells physically interacts with the strong tumour supressor BRCA2. Yeast and the generated intrabodies are thus expected to offer a useful system for studies on Sem1p/DSS1 function. Sem1p-specific antibody isolated from a phage display library was expressed intracellularily and targeted to either the cytosol or the nucleus of yeast cells. Analysis of the applicability of different antibody fragments as intrabodies showed that the Fab intrabody was expressed most efficiently. Expression of nuclear-targeted anti-Sem1p Fab intrabodies inhibited the growth of the sigma1278b yeast strain in a manner similar to deletion of the SEM1 gene. This indicates that the Fab intrabodies interact in vivo with Sem1p and result in inactivation of Sem1p. Localization of the Fab intrabody with or without the nuclear localization signal to the nucleus in Sem1p-dependent manner suggests that Sem1p mediates the nuclear transport of the intrabody without any targeting signal. Our results suggest that Sem1p function in yeast cells is in part manifested in the nucleus.

Amino Acid Sequence↗
PubMed2003

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries.

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)-hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP-hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.

Amino Acid Sequence↗
PubMed2002

Smart nanotubes for bioseparations and biocatalysis.

Tube-shaped nanostructures (nanotubes) have a number of attributes that make them potentially useful for biomedical applications such as drug delivery/detoxification and enzyme immobilization. Template synthesis provides a route for preparing monodisperse nanotubes of nearly any size and composed of nearly any material. We show here that template-synthesized silica nanotubes can be biochemically functionalized such that they act as biocatalysts and highly selective nano-phase extraction agents for bioseparations. For example, nanotubes containing an enantioselective antibody selectively extract the enantiomer of a drug molecule that binds to the antibody, relative to the enantiomer that has no specific interaction with the antibody. Nanotubes containing the enzyme glucose oxidase function as nanophase bioreactors to catalyze the oxidation of glucose.

Antibodies↗
PubMed2002

Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains.

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.

Amino Acid Sequence↗
PubMed2002

Antibody-based bio-nanotube membranes for enantiomeric drug separations.

Synthetic bio-nanotube membranes were developed and used to separate two enantiomers of a chiral drug. These membranes are based on alumina films that have cylindrical pores with monodisperse nanoscopic diameters (for example, 20 nanometers). Silica nanotubes were chemically synthesized within the pores of these films, and an antibody that selectively binds one of the enantiomers of the drug was attached to the inner walls of the silica nanotubes. These membranes selectively transport the enantiomer that specifically binds to the antibody, relative to the enantiomer that has lower affinity for the antibody. The solvent dimethyl sulfoxide was used to tune the antibody binding affinity. The enantiomeric selectivity coefficient increases as the inside diameter of the silica nanotubes decreases.

Aluminum Oxide↗
PubMed2002

Assigning probes into a small number of pools separable by electrophoresis.

MOTIVATION: Measuring transcriptional expression levels (transcriptional profiling) has become one of the most important methods in functional genomics. Still, new measuring methods are needed to obtain more reliable, quantitative data about transcription on a genomic scale. In this paper we concentrate on certain computational optimization problems arising in the design of one such novel method. From a computational point of view the key feature of the new method is that the hybridized probes are distinguished from each other based on their different size. Therefore the probes have to be assigned into pools such that the probes in the same pool have unique sizes different enough from each other. Identification of expressed RNA is given by probe pool and probe size while quantification is given by the label of the probe, e.g. fluorescence intensity. RESULTS: We show how to computationally find the probes and assign them into pools for a whole genome such that (i) each gene has a specific probe suitable for amplification and hybridization, and (ii) the expression level measurement can be done in a minimal number of pools separable by electrophoresis in order to minimize the total experiment cost of the measurement. Our main result is a polynomial-time approximation algorithm for assigning the probes into pools. We demonstrate the feasibility of the procedure by selecting probes for the yeast genome and assigning them into less than 100 pools. The probe sequences and their assignment into pools are available for academic research on request from the authors.

Algorithms↗