| 1. |
- Tegel, Hanna, et al.
(författare)
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High-throughput protein production--lessons from scaling up from 10 to 288 recombinant proteins per week
- 2009
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 4:1, s. 51-57
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Tidskriftsartikel (refereegranskat)abstract
- The demand for high-throughput recombinant protein production has markedly increased with the increased activity in the field of proteomics. Within the Human Protein Atlas project recombinantly produced human protein fragments are used for antibody production. Here we describe how the protein expression and purification protocol has been optimized in the project to allow for han- dling of nearly 300 different proteins per week. The number of manual handling steps has been significantly reduced (from 18 to 9) and the protein purification has been completely automated.
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| 2. |
- Tegel, Hanna, et al.
(författare)
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Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)
- 2010
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 69:2, s. 159-167
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Tidskriftsartikel (refereegranskat)abstract
- The effect of two Escherichia coli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).
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| 3. |
- Uhlén, Mathias, et al.
(författare)
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A human protein atlas for normal and cancer tissues based on antibody proteomics
- 2005
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 4:12, s. 1920-1932
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.
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