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High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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1996 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 7, no 2, 131-41 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
1996. Vol. 7, no 2, 131-41 p.
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-13472PubMedID: 8616269OAI: oai:DiVA.org:kth-13472DiVA: diva2:325481
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Structure-function analysis of insuline-like growth factor I interactions
Open this publication in new window or tab >>Structure-function analysis of insuline-like growth factor I interactions
1997 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 1997. 69 p.
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-2490 (URN)91-7170-150-8 (ISBN)
Public defence
1997-04-01, 00:00
Note
QC 20100618Available from: 2000-01-01 Created: 2000-01-01 Last updated: 2011-11-08Bibliographically approved

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