Recombinant Fusion Proteins for the Industrial Production of Disulfide Bridge Containing Peptides: Purification, Oxidation without Concatamer Formation, and Selective Cleavage

• Published in: Protein expression and purification Vol. 12; no. 3; pp. 404 - 414
• Main Authors: Döbeli, Heinz, Andres, Herbert, Breyer, Nicola, Draeger, Nicholas, Sizmann, Dorothea, Zuber, Maria Tomás, Weinert, Brian, Wipf, Beat
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.1998
• Subjects: AIDS/HIV; Amino Acid Sequence; Chromatography, High Pressure Liquid; Cyanogen Bromide - chemistry; Disulfides - chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gene Expression Regulation, Viral - genetics; HIV Envelope Protein gp41 - chemistry; HIV Envelope Protein gp41 - genetics; HIV Envelope Protein gp41 - immunology; HIV Envelope Protein gp41 - isolation & purification; HIV-1 - chemistry; HIV-1 - genetics; HIV-1 - immunology; human immunodeficiency virus (HIV); Humans; Immunodominant Epitopes - chemistry; Immunodominant Epitopes - genetics; Immunodominant Epitopes - immunology; Immunodominant Epitopes - isolation & purification; Mass Spectrometry; Molecular Sequence Data; Oxidation-Reduction; Peptides - chemistry; Peptides - genetics; Peptides - immunology; Peptides - isolation & purification; Protein Folding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; selective cleavage of recombinant fusion proteins; suppression of concatamer formation; human immunodeficiency virus (HIV); selective cleavage of recombinant fusion proteins; suppression of concatamer formation
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1997.0844

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 inEscherichia colito 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.