A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: an application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C.
A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30-100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni(2+)-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with 15N and/or 13C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.
Kohno
T
T
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kusunoki
H
H
Sato
K
K
Wakamatsu
K
K
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D006639:Histidine; D036341:Intercellular Signaling Peptides and Proteins; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014452:Ubiquitins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014862:Wasp Venoms
D002247:Carbon Isotopes; D017931:DNA Primers; D036341:Intercellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D014862:Wasp Venoms; D006639:Histidine; C071123:mastoparan X