Search

Somatostatin/growth hormone-inhibiting hormone is the peptide that inhibits secretion of somatotropin/growth hormone. Solid-phase synthesis methods are being currently used to produce somatostatin. Recombinant peptide synthesis is widely described for the production of small proteins and peptides; however, the production at industrial scale of peptides for biopharmaceutical applications is limited for economic reasons. Here, we propose the use of a new pGB-SMT plasmid to produce Somatostatin, as a C-terminal fusion protein with a Kluyveromyces lactis β-galactosidase fragment. To facilitate removal of that fragment by CNBr cleavage, a methionine residue was introduced at the N-terminal of the hormone peptide. The use of this construction enables an IPTG-free expression system. The suitability of this procedure has been assessed in a 15 l scale-up experiment yielding almost 300 mg, with purity >99 % and it is being implemented for commercial scale. The plasmid pGB-SMT here described is an alternative option for a cheap and high expression of other short peptide hormones. [ABSTRACT FROM AUTHOR]

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters. [ABSTRACT FROM AUTHOR]

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding. [ABSTRACT FROM AUTHOR]