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High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli

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Summary

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 13C6-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 13C6-glucose usage. Incorporation levels of 13C and/or 15N isotopes in purified 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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Abbreviations

BPTI:

bovine pancreatic trypsin inhibitor

DTT:

dithiothreitol

Gdn-HCl:

guanidinium hydrochloride

IAA:

β-indole acrylic acid

IGF-II:

insulin-like growth factor II

PBS:

phosphate-buffered saline

PDMS:

plasma desorption mass spectrometry

PFPA:

pentafluoro propionic acid

RP-HPLC:

reversed-phase high performance liquid chromatography

Z:

IgG-binding protein domain derived from staphylococcal protein A.

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Authors and Affiliations

  1. Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden

    Magnus Jansson & Lena Jendeberg

  2. Department of Structural Biochemistry, Biopharmaceuticals, Pharmacia AB, S-112 87, Stockholm, Sweden

    Magnus Jansson, Lena Jendeberg & Björn Nilsson

  3. Cener for Advanced Biotechnology and Medicine, Rugers University, 679 Hoes Lane, 08854-5638, Piscataway, NJ, USA

    Yu-Chin Li, Stephen Anderson & Gaetano T. Montelione

  4. Department of Molecular Biology and Biochemistry, Rugers University, 679 Hoes Lane, 08854-5638, Piscataway, NJ, USA

    Stephen Anderson & Gaetano T. Montelione

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  1. Magnus Jansson
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  2. Yu-Chin Li
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  5. Gaetano T. Montelione
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Jansson, M., Li, YC., Jendeberg, L. et al. High-level production of uniformly 15N-and 13C-enriched fusion proteins in Escherichia coli . J Biomol NMR 7, 131–141 (1996). https://doi.org/10.1007/BF00203823

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