(729b) Intein-Mediated One-Step Purification of Escherichia Coli Secreted Human Antibody Fragments | AIChE

(729b) Intein-Mediated One-Step Purification of Escherichia Coli Secreted Human Antibody Fragments


Coolbaugh, M. J. - Presenter, The Ohio State University
Wood, D. W. - Presenter, The Ohio State University
Miller, K. - Presenter, Pacific Northwest National Laboratory
Wu, W. - Presenter, Princeton University

The purification of proteins using self-cleaving affinity purification tags based on engineered inteins is an attractive alternative to conventional purification methods, including purification with His-6 tags followed by protease addition to remove the tag.  Currently, self-cleaving purification tag technology is generally limited to expression in prokaryotic hosts such as Escherichia coli. One of the major limitations of using E. coli as an expression host is that it is generally not capable of producing disulfide-bond containing proteins, such as antibodies and antibody fragments, in a soluble form.  The current study overcomes this limitation by secreting fusion proteins into the periplasm, where correct disulfide bond formation can occur. The target proteins used in this study were E. coli maltose-binding protein (MBP) and β-lactamase, as well as two disulfide-bond containing human antibody fragments. Each target protein of interest was fused downstream from a chitin-binding-domain/intein tag.  Secretion of these tag-intein-fusion proteins into the periplasm was accomplished by engineering a PelB secretion leader sequence upstream of the intein-tagged fusion gene.  After expression, the fusion proteins were recovered in active form from the periplasm through simple osmotic shock of cell culture.  A chitin-agarose affinity resin column purified the full-length tagged fusion protein from the rest of the periplasmic proteome. Induction of intein cleavage with a simple pH shift (from pH 8.5 to 6.5 at room temperature) released the purified target protein from the column.  In all cases, the purified proteins were highly active and obtained at high yield in soluble form, without refolding.  This work demonstrates the feasibility of coupling the ΔI-CM intein based self-cleaving tags with periplasmic secretion in E. coli to greatly increase the diversity and complexity of proteins that can be purified using this system, including proteins with structurally important disulfide linkages.