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High-level producti...
High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli.
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- Jansson, Magnus (författare)
- KTH,Bioteknologi
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Li, Y C (författare)
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- Jendeberg, Lena (författare)
- KTH,Bioteknologi
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Anderson, S (författare)
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Montelione, B T (författare)
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Nilsson, B (författare)
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(creator_code:org_t)
- 1996
- 1996
- Engelska.
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Ingår i: Journal of Biomolecular NMR. - 0925-2738 .- 1573-5001. ; 7:2, s. 131-41
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Nyckelord
- TECHNOLOGY
- TEKNIKVETENSKAP
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- ref (ämneskategori)
- art (ämneskategori)
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