| 1. |
- Hall, Per, 1954, et al.
(författare)
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Dissolved organic matter in abyssal sediments: Core recovery artifacts
- 2007
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Ingår i: Limnology and Oceanography. - 0024-3590. ; 52:1, s. 19-31
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Tidskriftsartikel (refereegranskat)abstract
- We report measurements of pore-water dissolved organic carbon (DOC), dissolved organic nitrogen, total dissolved carbohydrates, dissolved free monosaccharides, and ammonium in recovered deep-sea sediments from the Porcupine Abyssal Plain (PAP), Northeast Atlantic. There were distinct maxima close to the sediment–water interface of these constituents at all times of the year. The very high diffusive effluxes calculated from these porewater distributions were not compatible with simultaneous sediment trap measurements of particulate organic carbon, nitrogen, and carbohydrate fluxes toward the seafloor. Effluxes calculated from pore-water DOC distributions in recovered cores from another Atlantic deep-sea site, showing almost identical maxima as those at PAP, were more than an order of magnitude greater than simultaneous in situ chamber DOC flux measurements. We suggest that the dissolved organic matter maxima are predominantly artifacts induced by lysis of, or leakage from, mainly bacterial biomass resulting from decompression and/or warming during recovery of the sediment cores from the abyssal seafloor. Temperature elevation during core recovery from the abyss gives a N2 saturation of about 150%, and the combined effect of warming and decompression results in a CO2 saturation of about 135%, which together plausibly are associated with bubble formation creating cell bursting. Previous estimates of microbial biomass in abyssal sediments may be underestimates because of the difficulty of counting lysed bacterial cells. Since exoenzymes are inducible, previous measurements of their activities in recovered abyssal sediments may be overestimates.
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| 2. |
- Kronqvist, Nina, et al.
(författare)
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A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
- 2008
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 21:4, s. 247-255
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 109 variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (106 variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.
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| 3. |
- Kronqvist, Nina, et al.
(författare)
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Simplified characterization through site-specific protease-mediated release of affinity proteins selected by staphylococcal display
- 2008
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 278:1, s. 128-136
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Tidskriftsartikel (refereegranskat)abstract
- The production of candidate affinity proteins in a soluble form, for downstream characterization, is often a time-consuming step in combinatorial protein engineering methods. Here, a novel approach for efficient production of candidate clones is described based on direct cleavage of the affinity protein from the surface of Staphylococcus carnosus, followed by affinity purification. To find a suitable strategy, three new fusion protein constructs were created, introducing a protease site for specific cleavage and purification tags for affinity chromatography purifications into the staphylococcal display vector. The three modified strains were evaluated in terms of transformation frequency, surface expression level and protease cleavage efficiency. A protocol for efficient affinity purification of protease-released affinity proteins using the introduced fusion-tags was successfully used, and the functionality of protease-treated and purified proteins was verified in a biosensor assay. To evaluate the devised method, a previously selected HER2-specific affibody was produced applying the new principle and was used to analyze HER2 expression on human breast cancer cells.
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| 4. |
- Löfblom, John, et al.
(författare)
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Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications
- 2007
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 73:21, s. 6714-6721
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Tidskriftsartikel (refereegranskat)abstract
- For efficient generation of high-affinity protein-based binding molecules, fast and reliable downstream characterization platforms are needed. In this work, we have explored the use of staphylococcal cell surface display together with How cytometry for affinity characterization of candidate affibody molecules directly on the cell surface. A model system comprising three closely related affibody molecules with different affinities for immunoglobulin G and an albumin binding domain with affinity for human serum albumin was used to investigate advantages and differences compared to biosensor technology in a side-by-side manner. Equilibrium dissociation constant (K-D) determinations as well as dissociation rate analysis were performed using both methods, and the results show that the on-cell determinations give both KD and dissociation rate values in a very fast and reproducible manner and that the relative affinities are very similar to the biosensor results. Interestingly, the results also show that there are differences between the absolute affinities determined with the two different technologies, and possible explanations for this are discussed. This work demonstrates the advantages of cell surface display for directed evolution of affinity proteins in terms of fast postselectional, on-cell characterization of candidate clones without the need for subcloning and subsequent protein expression and purification but also demonstrates that it is important to be aware that absolute affinities determined using different methods often vary substantially and that such comparisons therefore could be difficult.
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| 5. |
- Löfblom, John, et al.
(författare)
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Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display
- 2005
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 248:2, s. 189-198
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated a staphylococcal surface display system for its potential future use as a protein library display system ill combinatorial biochemistry. Efficient affinity-based selections require a system capable of fine affinity discrimination of closely related binders to minimize the loss of potentially improved variants. In this Study, a significant breakthrough was achieved to avoid biases due to potential cell-to-cell variations in surface expression levels, since it was found that a generic protein tag, present within the displayed recombinant surface proteins on the cells, could be successfully employed to obtain normalization of the target-binding signal. Four mutated variants of a staphylococcal protein A domain with different affinity to human IgG were successfully expressed on the surface of recombinant Staphylococcus carnosus cells. The system was evaluated for affinity-based cell sorting experiments, where cell-displayed protein A domains with an 8-fold difference in target affinity were mixed at a ratio of 1: 1000 and sorted using FACS. Enrichment factors around 140-fold were obtained from a single round of sorting under normal library sorting conditions when the top 0.1% fraction having the highest antigen binding to Surface expression level ratio was sorted. The results demonstrate that the system would have a potential as a selection system in protein library display applications, and the normalization strategy should indeed make it possible to achieve fine affinity discriminations in future library selections. (c) 2005 Federation of European Microbiological Societies.
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| 6. |
- Löfblom, John, et al.
(författare)
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Optimization of electroporation-mediated transformation : Staphylococcus carnosus as model organism
- 2007
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Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 102:3, s. 736-747
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Tidskriftsartikel (refereegranskat)abstract
- The study was conducted with an aim to optimize the transformation efficiency of the Gram-positive bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface displayed combinatorial protein libraries. Methods and Results: We have thoroughly investigated a number of different parameters for: (i) the preparation of electrocompetent cells; (ii) the treatment of cells before electroporation; (iii) the electroporation step itself; and (iv) improved recovery of transformed cells. Furthermore, a method for heat-induced inactivation of the host cell restriction system was devised to allow efficient transformation of the staphylococci with DNA prepared from other species, such as Escherichia coli. Previously described protocols for S. carnosus, giving transformation frequencies of approximately 10(2) transformants per transformation could be improved to reproducible procedures giving around 10(6) transformants for a single electroporation event, using plasmid DNA prepared from either S. carnosus or E. coli. The transformed staphylococcal cells were analysed using flow cytometry to verify that the entire cell population retained the introduced plasmid DNA and expressed the recombinant protein in a functional form on the cell surface at the same level as the positive control population. Conclusions: The results demonstrate that the transformation frequency for S. carnosus could be dramatically increased through optimization of the entire electroporation process, and that the restriction barrier for interspecies DNA transfer, could be inactivated by heat treatment of the cells prior to electroporation. Significance and Impact of the Study: The generation of large combinatorial protein libraries, displayed on the surface of S. carnosus can be envisioned in the near future, thus dramatically improving the selection compared with the traditional biopanning procedure used in phage display.
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| 7. |
- Löfblom, John, 1977-
(författare)
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Staphylococcal surface display for protein engineering and characterization
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution. Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering. In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins. Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization.
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| 8. |
- Sandin, Gunnar, et al.
(författare)
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Feedback: an Architectural Interpretation of a Painting by Meret Opppenheim
- 2009
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Ingår i: La Semiotica. Intersección entre la Naturaleza y la Cultura / Semiotics Bridging Nature and Culture / La sémotique: carrefour de la nature et de la culture. : Proceedings of the 6th Congress of the IASS-AIS, Guadalajara, Mexico, 13-19 July, 1999. - Proceedings of the 6th Congress of the IASS-AIS, Guadalajara, Mexico, 13-19 July, 1999..
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Konferensbidrag (refereegranskat)
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| 9. |
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| 10. |
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| 11. |
- Ståhl, Anne-Lie, et al.
(författare)
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Factor H dysfunction in patients with atypical hemolytic uremic syndrome contributes to complement deposition on platelets and their activation.
- 2008
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 111, s. 5307-5315
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Tidskriftsartikel (refereegranskat)abstract
- Atypical hemolytic uremic syndrome (aHUS) may be associated with mutations at the C-terminal of factor H (FH). FH binds to platelets via the C-terminal as previously shown using a construct consisting of short consensus repeats (SCRs) 15-20. Four FH mutations, in SCR15 (C870R) and SCR20 (V1168E, E1198K, E1198Stop) in aHUS patients, were studied regarding their ability to allow complement activation on platelet surfaces. Purified FH-E1198Stop mutant exhibited reduced binding to normal washed platelets compared to normal FH, detected by flow cytometry. Washed platelets taken from the four aHUS patients during remission exhibited C3 and C9 deposition, as well as CD40-ligand (CD40L) expression indicating platelet activation. Combining patient serum/plasma with normal washed platelets led to C3 and C9 deposition, CD40L and CD62P expression, aggregate formation and generation of tissue factor-expressing microparticles. Complement deposition and platelet activation were reduced when normal FH was pre-incubated with platelets and were minimal when using normal serum. The purified FH-E1198Stop mutant added to FH-deficient plasma (complemented with C3) allowed considerable C3 deposition on washed platelets, in comparison to normal FH. In summary, mutated FH enables complement activation on the surface of platelets and their activation, which may contribute to the development of thrombocytopenia in aHUS.
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| 12. |
- Ståhl, Annelie, et al.
(författare)
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Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different
- 2005
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 349:4, s. 847-860
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Tidskriftsartikel (refereegranskat)abstract
- Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P′1P′1position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10–16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10–23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.
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| 13. |
- Ståhl, Lars-Henrik, et al.
(författare)
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Diagram och projektioner
- 2006
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Ingår i: Utforskande Arkitektur. - 9197590142
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 14. |
- Ståhl, Lars-Henrik
(författare)
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Fast Food, Language… Architecture
- 2005
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Ingår i: 01.AKAD beginnings: experimental research in architecture and design 01.AKAD startpunkter : experimentell forskning inom arkitektur och design. - 919759010X
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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