A fusion protein system for the recombinant production of short disulfide-containing peptides.
A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000378:metabolism; D003500:Cyclization; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004220:Disulfides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003488:Cyanogen Bromide