title: Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage.
abstract: We report the biotechnical production of peptides of approximately 35-50 amino acids in length containing one intramolecular disulfide bridge, using a recombinant fusion tail approach. This method fills the technological gap when either (a) chemical synthesis fails due to known problematic peptide sequences or (b) if simple recombinant expression is unsuccessful due to degradation. The fusion tail described here serves several purposes: (i) it enables high expression levels in Escherichia coli to be achieved; (ii) it renders the fusion protein fairly soluble; (iii) it contains a histidine affinity tag for easy purification on Ni-chelate resins, which also serves as a catalyst for the oxygen-dependent formation of the disulfide bridge; and (iv) it suppresses the formation of concatamers during the oxidation process through steric hindrance. The purified fusion protein is then immobilized on a reversed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the peptide. A very hydrophilic fusion partner is required so that immobilization on the reversed phase column always occurs due to the peptide. Sensitive hydrophobic residues are thereby protected from the cleavage reagent while the cleaved hydrophilic fusion tail is easily separated from the hydrophobic peptide. The method is exemplified by eight peptides representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptides.
authors.affiliation: Preclinical Research, F. Hoffmann-La Roche, Basel, CH-4070, Switzerland.
authors.forename: H
authors.initials: H
authors.lastname: Döbeli
authors.forename: H
authors.initials: H
authors.lastname: Andres
authors.forename: N
authors.initials: N
authors.lastname: Breyer
authors.forename: N
authors.initials: N
authors.lastname: Draeger
authors.forename: D
authors.initials: D
authors.lastname: Sizmann
authors.forename: M T
authors.initials: MT
authors.lastname: Zuber
authors.forename: B
authors.initials: B
authors.lastname: Weinert
authors.forename: B
authors.initials: B
authors.lastname: Wipf
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D004220:Disulfides; D015700:HIV Envelope Protein gp41; D016056:Immunodominant Epitopes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003488:Cyanogen Bromide