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Overview of the Purification of Recombinant Proteins Produced in Escherichia coli

Paul T. Wingfield¹

¹National Institutes of Health, Bethesda, Maryland

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 6.1

DOI:

10.1002/0471140864.ps0601s30

Online Posting Date:

February, 2003

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Paul Wingfield

Abstract

The updated version of this unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. coli. The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a discussion of common problems associated with bacterial protein expression. A flow chart summarizes approaches for establishing solubility and localization of bacterially produced proteins. Purification strategies for both soluble and insoluble proteins are also reviewed. A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. Finally, protein handling, scale and aims of purification, and specialized equipment needed for recombinant protein purification and characterization are discussed. The methodologies and approaches described here are essentially suitable for laboratory-scale operations.

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Unit Introduction
Protein Sequence and Compositional Analysis
Characteristics of the Host-Vector System
Solubility and Location of the Protein
Strategies for Isolation of Soluble Proteins
Strategies for Isolation of Insoluble Proteins
Bacterial Expression of Proteins Normally Glycosylated
Some Examples of Protein Expression and Purification
Protein Handling
Scale of Operations and Aims of Purification
Specialized Equipment
Literature Cited
Figures
Tables

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Figures

Figure 6.1.1

Differential centrifugation of E. coli cell lysates. Cells are broken with a French press or by lysozyme treatment. Insoluble (inclusion body) proteins, from either the cytoplasm or periplasm, are located in the low-speed pellet, which is subjected to preextraction to remove outer membrane and peptidoglycan material. Inclusion bodies are extracted from washed pellets with strong protein denaturants such as guanidine·HCl. The solubilized protein, which is denatured and reduced (free sulfhydryl residues), is either directly folded and oxidized (disulfide bonds formed) or purified before folding. Soluble proteins (from the periplasm and cytoplasm) are located in the low-speed and high-speed supernatants. The latter can be used directly for chromatography, whereas the former requires clarification by other techniques such as ammonium sulfate fractionation or membrane filtration.

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Figure 6.1.2

Localization of secreted and periplasmic proteins in E. coli. Periplasmic protein produced via a secretion vector can leak into the medium and be recovered by centrifugation (supernatant, S1) or filtration. Washing cells with an isotonic solution such as lightly buffered 0.15 M NaCl or 0.25 M sucrose can also release protein (S2). The compartmentalized periplasmic proteins are released by isotonic shock treatment by directly suspending normal cell paste or plasmolyzed cell paste into hypotonic medium. Plasmolyzed cell paste is derived by suspending cells in hypertonic medium and then pelleting. (In hypertonic medium the cell contracts, separating the inner membrane from the cell wall, and is said to be osmotically sensitized.) The hypertonic wash often releases protein (P1). The supernatant from shocked cells (P2) will contain constitutive E. coli proteins and the recombinant product. Osmotically sensitized cells can also be treated with lysozyme to fragment the outer membrane, thus releasing periplasmic proteins (P3). The pellet from the lysozyme treatment contains spheroplasts (cells with fragmented outer membranes), which are easily disrupted by detergents, sonication, or hypotonic shock to release cytoplasmic proteins.

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Figure 6.1.3

Purification of soluble proteins from E. coli lysates. Abbreviations for ion-exchange resins are as follows: CM, carboxymethyl; DEAE, diethylaminoethyl; Q, quaternary ammonium; S, methyl sulfonate. The order of preference for the stages of ion-exchange (2) and other methods (3) is based on the author's opinion and does not necessarily represent a consensus view. On the other hand, the use of a DEAE-based matrix at an early stage (1) is common practice. Affinity methods (see text and Chapter 9) can be performed at any stage following clarification of the lysate.

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Figure 6.1.4

Folding and purification of inclusion body proteins from E. coli. The protein is extracted with protein denaturants such as guanidine·HCl (Gu·HCl), urea, or an organic acid. The reductant dithiothreitol (DTT) is included to prevent artificial disulfide bond formation (especially intermolecular bonds). The denatured protein can be purified by various methods and then folded, or it can be directly folded. Typically, some purification (e.g., gel filtration in Gu·HCl) prior to folding is recommended as it often results in higher folding yields. Protein folding and oxidation are carried out concurrently. Disulfide bond formation is catalyzed by low-molecular-weight thiol/disulfide pairs such as reduced (GSH) and oxidized (GSSG) glutathione. GSH/GSSG ratios of 5:1 to 10:1 are normally used, which are similar to those found in vivo in the endoplasmic reticulum (Hwang et al., 1992). A cosolvent is included to maintain solubility during folding. Folded protein is purified if necessary (purification is usually needed if the protein is directly folded). Gel filtration is a useful final step for removing aggregated and or misfolded protein.

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Figure 6.1.5

Preparation of washed pellets using lysozyme and the French press. Cells are broken with the French press with or without prior treatment with lysozyme. After low-speed centrifugation using a fixed-angle rotor, the contents of the centrifuge tubes have the characteristics shown. The contents of tubes A and B are labeled: s, supernatant; lp, loose pellet; ib, inclusion body protein; and c, unbroken cells and large cellular debris. The loose pellet material is derived from the outer cell wall and outer membrane (see text for further details). After washing the insoluble material (unit 6.3), the pellet should consist mainly of the inclusion body layer (tube C), and the supernatant should be fairly clear.

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Author Notes

Submitted by: Paul Wingfield

On: August 17, 2009

Most recombinant protein purifications are now performed by incorporating affinity purification tags the most popular of which is the His-tag. Several venders sell the Ni -affinity media used to purify the tagged proteins and it is worth while to occasionally check their respective web sites for improved media and their characteristics. The various media differ in their tolerance, for example, of reducing reagents and anionic detergents. One important fact to bear in mind is that the His-tag is not inert and will bind among other things, lipopolysaccharides (LPS) and nucleic acids, so for biological testing its not a bad idea to remove the tag.

In the unit the expression of soluble vs. insoluble proteins may be appear to be clear cut whereas in reality the expression of many recombinant proteins are mixed For example, proteins and protein domains which in their native state interact with other proteins, these proteins will often be distributed between insoluble and soluble phases of the cell lysates. To avoid problems in purification due to the tendency of these proteins to bind to host proteins and other cellular components, it is often best to purify them in presence of denaturants and then fold after purification.

Submitted by: Paul Wingfield

On: August 17, 2009

Readers may be interested in "Protein production and purification" Nature Methods (2008) 5: 135-146. This is a summary of methods used from various structural genomic consortiums.