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

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

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

Protein Expr Purif. 2014 Mar 31;

Authors: Nespovitaya N, Barylyuk K, Eichmann C, Zenobi R, Riek R

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 His6-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 (13)C and (15)N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.


PMID: 24698890 [PubMed - as supplied by publisher]



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