Peptide affinity chromatography media that bind N(pro) fusion proteins under chaotropic conditions.

Rainer Hahn, Michael Seifert, Sabine Greinstetter, Barbara Kanatschnig, Eva Berger, Waltraud Kaar, Alois Jungbauer

Index: J. Chromatogr. A. 1217(40) , 6203-13, (2010)

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Abstract

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N(pro) fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N(pro) from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7-15 mg protein mL(-1) of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.Copyright © 2010 Elsevier B.V. All rights reserved.