RECOMBINANT FUSION PROTEINS FOR THE INDUSTRIAL-PRODUCTION OF DISULFIDE BRIDGE CONTAINING PEPTIDES - PURIFICATION, OXIDATION WITHOUT CONCATAMER FORMATION, AND SELECTIVE CLEAVAGE

We report the biotechnical production of peptides of approximately 35- 50 amino acids in length containing one intramolecular disulfide bridg e, using a recombinant fusion tail approach. This method fills the tec hnological gap when either (a) chemical synthesis fails due to known p roblematic peptide sequences or (b) if simple recombinant expression i s unsuccessful due to degradation. The fusion tail described here serv es several purposes: (i) it enables high expression levels in Escheric hia coli to be achieved; (ii) it renders the fusion protein fairly sol uble; (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 th e formation of concatamers during the oxidation process through steric hindrance. The purified fusion protein is then immobilized on a rever sed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the pept ide. A very hydrophilic fusion partner is required so that immobilizat ion on the reversed phase column always occurs due to the peptide. Sen sitive hydrophobic residues are thereby protected from the cleavage re agent while the cleaved hydrophilic fusion tail is easily separated fr om the hydrophobic peptide. The method is exemplified by eight peptide s representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptide s. (C) 1998 Academic Press.