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

J Biomol NMR. 1996 Mar;7(2):131-41. doi: 10.1007/BF00203823.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aprotinin / biosynthesis
  • Aprotinin / genetics
  • Carbon Isotopes
  • Cattle
  • Escherichia coli / metabolism*
  • Humans
  • Insulin-Like Growth Factor II / biosynthesis
  • Insulin-Like Growth Factor II / genetics
  • Isotope Labeling / methods*
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Nitrogen Isotopes
  • Recombinant Fusion Proteins / biosynthesis*
  • Staphylococcal Protein A / biosynthesis
  • Staphylococcal Protein A / genetics


  • Carbon Isotopes
  • Nitrogen Isotopes
  • Recombinant Fusion Proteins
  • Staphylococcal Protein A
  • Insulin-Like Growth Factor II
  • Aprotinin