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Protein Purification: Theoretical and Methodological Considerations

Peptides and Proteins

  1. Béla János Takács

Published Online: 15 SEP 2006

DOI: 10.1002/9780470027318.a1626m

Encyclopedia of Analytical Chemistry

Encyclopedia of Analytical Chemistry

How to Cite

Takács, B. J. 2006. Protein Purification: Theoretical and Methodological Considerations. Encyclopedia of Analytical Chemistry. .

Author Information

  1. F. Hoffmann-La Roche Ltd, Basel, Switzerland

Publication History

  1. Published Online: 15 SEP 2006

Abstract

With the rapid advances in recombinant DNA technologies, it became possible to express large amounts of virtually any protein in different host cells, including bacteria, yeast, insect, and mammalian cells. Efficient purification methods are becoming more and more important as the need for protein drugs continues to expand. In this article we cover some basic procedures for the purification of soluble recombinant proteins expressed in Escherichia coli and methods to obtain soluble and active protein from insoluble and inactive inclusion bodies. Finally, methods for the purification of native intrinsic membrane proteins from human lymphoblastoid cell lines, using immobilized monoclonal antibodies (mAbs), are presented.

As a first step, the protein of interest has to be released into an aqueous extract. Following extraction, residual debris and intact cells are removed by centrifugation. Nucleic acids are either hydrolyzed or precipitated to reduce the viscosity of the extract. A crude enrichment for the protein of interest is made based on its differential solubility in ammonium sulfate. This is then followed by high-resolution chromatographic methods. The type of chromatographic matrices and the order of their use are evaluated in pilot experiments for column selection. By combining the specificity and discriminatory powers of various chromatographic techniques, it is possible to isolate rapidly a single protein species from a heterogeneous mixture.

For the purification of native intrinsic membrane proteins, the use of immunoaffinity methods is emphasized, since specific mAbs may be obtained even against low-copy-number membrane proteins by reducing a multispecific antibody response to a series of monospecific responses by cloning. The use of immobilized, highly specific mAbs is demonstrated in the purification of human leukocyte antigen D-related (HLA-DR) antigens.

Rapid advances in complete genome sequences provide us with a continuously expanding number of new potential therapeutic targets. These proteins will have to be purified and characterized. The existence of a functional protein blueprint will be invaluable not only for the diagnosis but possibly also for the treatment of many diseases.