The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.
Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer polypeptide product lowered the yield of the target peptide and complicated its purification. The side product contained the N-terminal 6His-tag together with the thioredoxin fusion partner and the specific enzymatic cleavage site-containing linker followed by an unknown peptide starting with the first 7N-terminal amino acid residues of SS14, as revealed by the Edman degradation. The combination of DNA sequence analysis, the Edman degradation, and high-resolution mass spectrometry allowed to identify the amino acid sequence of the unknown peptide. The appearance of the side product was attributed to inefficient termination of mRNA translation. The stop codon and its downstream sequence optimization allowed eliminating the side product synthesis. The optimized expression system, purification protocol, and post-translational modification procedure yielded 1.5mg of SS14 per liter of minimal medium. Nearly 99% incorporation of (13)C and (15)N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.
Laboratory of Physical Chemistry, HCI F223, Vladimir-Prelog-Weg 2, 8093 Zurich, Switzerland. Electronic address: nadezda.nespovitaya@phys.chem.ethz.ch.
Nadezhda
N
Nespovitaya
Laboratory of Organic Chemistry, HCI E325, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland. Electronic address: barylyuk@org.chem.ethz.ch.
Konstantin
K
Barylyuk
Laboratory of Physical Chemistry, HCI F223, Vladimir-Prelog-Weg 2, 8093 Zurich, Switzerland. Electronic address: cedric.eichmann@phys.chem.ethz.ch.
Cédric
C
Eichmann
Laboratory of Organic Chemistry, HCI E325, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland. Electronic address: zenobi@org.chem.ethz.ch.
Renato
R
Zenobi
Laboratory of Physical Chemistry, HCI F223, Vladimir-Prelog-Weg 2, 8093 Zurich, Switzerland. Electronic address: roland.riek@phys.chem.ethz.ch.
Roland
R
Riek
D002247:Carbon Isotopes; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013004:Somatostatin / Q000096:biosynthesis / Q000302:isolation & purification*
D002247:Carbon Isotopes; D018388:Codon, Terminator; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D013004:Somatostatin
Escherichia coli; Frameshift; Isotopic labelling; Oxidation; Purification; Somatostatin 14; Stop codon optimization; TAA stop codon