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 N-15 AND N-15 C-13/

A new strategy is described for the production of peptides enriched wi th 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 is otope-enriched fusion proteins in minimal media. A decahistidine tag a ttached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni2+-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a pro tease, 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 fus ion 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 c ells. As an example, the expression and the uniform stable isotope enr ichment with N-15 and/or C-13 are described for mastoparan-X, a tetrad ecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our met hod. The presented system is considered powerful for the stable isotop e enrichment of short peptides with proton resonances that are too sev erely overlapped to be analyzed solely by proton NMR.