High-level production of uniformly ¹⁵N- and ¹³C-enriched fusion proteins in Escherichia coli.
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.
Jansson
M
M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Li
Y C
YC
Jendeberg
L
L
Anderson
S
S
Montelione
G T
GT
Nilsson
B
B
D000818:Animals; D007611:Aprotinin / Q000096:biosynthesis / Q000235:genetics; D002247:Carbon Isotopes; D002417:Cattle; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000235:genetics
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D007335:Insulin-Like Growth Factor II; D007611:Aprotinin