| 1. |
- Ahmadian, Afshin, et al.
(författare)
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Pyrosequencing : History, biochemistry and future
- 2006
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Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981 .- 1873-3492. ; 363:02-jan, s. 83-94
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Forskningsöversikt (refereegranskat)abstract
- Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved. Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.
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| 2. |
- Alm, Tove, et al.
(författare)
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High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
- 2007
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 2, s. 709-716
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Tidskriftsartikel (refereegranskat)abstract
- A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.
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| 3. |
- Berglund, Lisa, et al.
(författare)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Forskningsöversikt (refereegranskat)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 4. |
- Björling, Erik, et al.
(författare)
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A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:5, s. 825-844
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Tidskriftsartikel (refereegranskat)abstract
- Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.
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| 5. |
- Boström, Maria, et al.
(författare)
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Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
- 2005
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 68:1, s. 82-90
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Tidskriftsartikel (refereegranskat)abstract
- The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.
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| 8. |
- Eriksson, Cecilia, 1977-
(författare)
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Affinity based proteomics research tools
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Listen to the mantra; the mapping of the genome was finished in 2001, and the sequel research challenge is the thorough survey of the corresponding human proteome. This was stated almost a decade ago, it has been repeated over and over, and is still most certainly a hard nut to crack. The workload is daunting, much because there is no protein amplification method and no binary system for the detection of proteins, and because the complexity of the proteome is larger than that of the genome as it seems. Hence, there is a need for high throughput technologies that, at sufficiently low limits of detection and with satisfying sensitivities, may investigate protein content in human samples. With this aim, the Human Proteome Resource (HPR) project was initiated in 2003. All work presented in this thesis relate to protein interactions; binders are either utilized such as for the depletion of high abundant proteins from serum, or analyzed such as in the validation of monospecific antibody specificity, or the epitope mapping of the same. In Paper I, the Gyrolab system is utilized in a setup for the specificity analysis of monospecific antibodies towards their antigen, and the setup is compared to planar protein arrays. Gyrolab technology is used again, in Paper II, where epitope mapping of monospecific antibodies is performed in order to analyze antibody specificity. Also, mapping serves to compare the immune-responses from parallel immunizations using the same antigen, thereby assessing reproducibility in the regeneration of antibodies. Paper III describes a high throughput approach for the depletion of high abundant proteins, in serum and plasma samples, taking advantage of Affibody molecules as binders. The last two papers, IV and V, utilize monospecific antibodies for protein analysis; in Paper IV pull out experiments show that competitive elution using the PrEST antigen can be a fruitful approach to increase specificity. And finally, in Paper V, a setup for the semi-quantitative protein content analysis in fluid samples is suggested. Again, the Gyrolab technology is used, and the setup is tested on a simplified model system.
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| 9. |
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| 10. |
- Eriksson, Cecilia, et al.
(författare)
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Microfluidic analysis of antibody specificity in a compact disk format
- 2006
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:7, s. 1568-1574
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Tidskriftsartikel (refereegranskat)abstract
- A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His(6)-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three- dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.
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