Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage.
Journal - Protein expression and purification (UNITED STATES )
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.Copyright 1998 Academic Press.
|ISSN : ||1046-5928|
|Mesh Heading : ||Amino Acid Sequence Chromatography, High Pressure Liquid Cyanogen Bromide Disulfides Electrophoresis, Polyacrylamide Gel Escherichia coli Gene Expression Regulation, Viral HIV Envelope Protein gp41 HIV-1 Humans Immunodominant Epitopes Mass Spectrometry Molecular Sequence Data Oxidation-Reduction Peptides Protein Folding Recombinant Fusion Proteins chemistry chemistry genetics genetics chemistry genetics immunology chemistry genetics chemistry genetics immunology chemistry genetics immunology chemistry genetics immunology|
|Mesh Heading Relevant : ||isolation & purification immunology isolation & purification isolation & purification isolation & purification|