Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage

Protein Expression and Purification
H DöbeliB Wipf

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 th...Continue Reading

Citations

Aug 9, 2002·Tropical Medicine & International Health : TM & IH·M KnappikT Jelinek
Dec 25, 2004·Biophysical Chemistry·Patricia T CampanaM Lucia Bianconi
Dec 7, 2006·Plant Biotechnology Journal·Andrea MolinaJon Veramendi
Dec 25, 2015·Fungal Genetics and Biology : FG & B·Fabiola PolliArnold J M Driessen
Jan 14, 2000·Nature Biotechnology·F Grüninger-LeitchH Döbeli
Aug 31, 2004·Biotechnology and Bioengineering·Giacomo MorrealeAnton P J Middelberg
Sep 10, 2003·Protein Expression and Purification·Richard J JennyRoger L Lundblad
Jan 13, 2005·Journal of Molecular Biology·Christian ScholzFranz Xaver Schmid
Jul 15, 2006·Protein Expression and Purification·Marzena Pazgier, Jacek Lubkowski
Jan 30, 2008·Protein Expression and Purification·De-ying MaAn-shan Shan

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