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

Magnus Jansson, Yu Chin Li, Lena Jendeberg, Stephen Anderson, Gaetano T. Montelione, Björn Nilsson

Research output: Contribution to journalArticlepeer-review

143 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)131-141
Number of pages11
JournalJournal of biomolecular NMR
Volume7
Issue number2
DOIs
StatePublished - 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Spectroscopy

Keywords

  • Biosynthetic enrichment
  • Bovine pancreatic trypsin inhibitor
  • Insulin-like growth factor
  • Staphylococcal protein A
  • Triple-resonance NMR

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