RESUMO
Neutron Therapy: An Experimental System in Radiooncology. The history of the use of neutrons in radiotherapy will be revisited by focusing on the ideas, theories and experiments that led to first clinical studies. For addressing epistemological questions regarding biological effects of fast neutrons, the notion of an "experimental system" is employed and its evolution over time discussed. Taking up the analytical framework of Hans-Jörg Rheinberger, biological effects and the physical instrument "cyclotron" are conceptualized in terms of epistemic and technical objects. It is shown that the epistemic object "biological effects of neutrons" could never reach the status of a technical object and - more strikingly - that the cyclotron itself returned to being an epistemic object in the context of medicine. So there is a misunderstanding of the different roles epistemic and technical objects have to play in the framework of an experimental system. This misunderstanding led to today's highly controversial discussions about the application of neutrons in radiotherapy.
Assuntos
Oncologia/história , Pesquisa/história , Alemanha , História do Século XIX , História do Século XX , Humanos , NeoplasiasRESUMO
BACKGROUND: An arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment. RESULTS: A robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25 degrees C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead. CONCLUSION: Soluble fusion proteins can be produced in E. coli in sufficient qualities and microg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.
RESUMO
Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.
