The production of recombinant 15N, 13C-labelled somatostatin 14 for NMR spectroscopy

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Author(s) Nespovitaya, Nadezhda, Barylyuk, Konstantin, Eichmann, Cédric, Zenobi, Renato, Riek, Roland
Publication Type Journal Items, Publication Status: Published
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Title The production of recombinant 15N, 13C-labelled somatostatin 14 for NMR spectroscopy
Author(s) Nespovitaya, Nadezhda
Barylyuk, Konstantin
Eichmann, Cédric
Zenobi, Renato
Riek, Roland
Journal or Series Title Protein expression & purification
Volume Number 99
Start Page 78
End Page 86
ISSN 1046-5928
Publisher Elsevier
Publication Place Amsterdam
Publication Date 2014-07
Keyword(s) Somatostatin 14
Escherichia coli
TAA stop codon
Stop codon optimization
Isotopic labelling
Abstract 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 7 N-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 1.5 mg of SS14 per liter of minimal medium. Nearly 99% incorporation of 13C and 15N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.
DOI 10.1016/j.pep.2014.03.011
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03782
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NEBIS System Number 000569627
Source Database ID FORM-1415358965
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  author = "Nespovitaya, Nadezhda and Barylyuk, Konstantin and Eichmann, C{\'{e}}dric and Zenobi, Renato and Riek, Roland",
  title = "{T}he production of recombinant 15{N}, 13{C}-labelled somatostatin 14 for {N}{M}{R} spectroscopy",
  journal = "Protein expression \& purification",
  year = 2014,
  volume = "99",
  pages = "78--86",
  month = jul,

E-Citations record created: Fri, 07 Nov 2014, 11:16:29 CET