Self-Immobilizing Recombinant Antibody Fragments for Immunoaffinity Chromatography: Generic, Parallel, and Scalable Protein Purification

Authors: Blank K.¹; Lindner P.¹; Diefenbach B.²; Plückthun A.¹

Source: Protein Expression and Purification, Volume 24, Number 2, March 2002 , pp. 313-322(10)

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Abstract:

We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin-binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins). Copyright 2002 Elsevier Science (USA).

Keywords: directed protein immobilization; recombinant antibody fragment; scFv; immunoaffinity chromatography; fusion protein; chitin-binding domain.

Language: English

Document Type: Research article

Affiliations: 1: Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland 2: MorphoSys AG, Lena-Christ-Strasse 48, Martinsried/Munich, D-82152, Germany

Publication date: 2002-03-01