| 2. |
- Forsström, Bjorn, et al.
(författare)
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Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
- 2014
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:6, s. 1585-1597
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.
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| 3. |
- Tegel, Hanna, et al.
(författare)
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High throughput generation of a resource of the human secretome in mammalian cells
- 2020
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 58, s. 45-54
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic beta-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.
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