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- Jansson, Magnus, et al.
(författare)
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High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli.
- 1996
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Ingår i: Journal of Biomolecular NMR. - 0925-2738 .- 1573-5001. ; 7:2, s. 131-41
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Tidskriftsartikel (refereegranskat)abstract
- An approach to produce 13C- and 15N-enriched proteins is described. The concept is based on intracellular production of the recombinant proteins in Escherichia coli as fusions to an IgG-binding domain, Z, derived from staphylococcal protein A. The production method provides yields of 40-200 mg/l of isotope-enriched fusion proteins in defined minimal media. In addition, the Z fusion partner facilitates the first purification step by IgG affinity chromatography. The production system is applied to isotope enrichment of human insulin-like growth factor II (IGF-II), bovine pancreatic trypsin inhibitor (BPTI), and Z itself. High levels of protein production are achieved in shaker flasks using totally defined minimal medium supplemented with 13C(6)-glucose and (15NH4)2SO4 as the only carbon and nitrogen sources. Growth conditions were optimized to obtain high protein production levels and high levels of isotope incorporation, while minimizing 13C(6)-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purifies IGF-II, BPTI, and Z were confirmed using mass spectrometry and NMR spectroscopy. More than 99% of total isotope enrichment was obtained using a defined isotope-enriched minimal medium. The optimized systems provide reliable, high-level production of isotope-enriched fusion proteins. They can be used to produce 20-40 mg/l of properly folded Z and BPTI proteins. The production system of recombinant BPTI is state-of-the-art and provides the highest known yield of native refolded BPTI.
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- Bentz, M, et al.
(författare)
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Chromosome imbalances in papillary renal cell carcinoma and first cytogenetic data of familial cases analyzed by comparative genomic hybridization
- 1996
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Ingår i: Cytogenetics and cell genetics. - : S. Karger AG. - 0301-0171. ; 75:1, s. 17-21
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Tidskriftsartikel (refereegranskat)abstract
- We used comparative genomic hybridization to analyze 17 tumor samples from 11 patients with papillary renal cell carcinoma (RCC), including three patients with hereditary papillary RCC. Whereas the most frequent aberrations confirmed data obtained by banding analyses, copy number increases on 5q, which previously were considered characteristic of nonpapillary RCC, were identified in two cases. In two complex cases belonging to the same family, a characteristic pattern of chromosomal aberrations was found: five of the six imbalances present in the less complex case were included in the karyotype of the other case, suggesting a genetically determined mechanism resulting in genomic instability of specific chromosomes or chromosomal subregions and/or selection of specific mutations.
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