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An Overview of Protein Purification Methods

A fundamental step in studying individual proteins is purification of the protein of interest. There are four basic steps of protein purification: 1 cell lysis, 2 protein binding to a matrix, 3 washing and 4 elution. Cell lysis can be accomplished a number of ways, including nonenzymatic methods e. Because purification of native proteins can be challenging, affinity purification tags are often fused to a recombinant protein of interest such that the tag is used to capture or detect the protein.

Here we provide an overview of protein purification strategies, including guidelines on choosing a purification method and example protocols for protein purification using affinity tags. Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction.

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Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications.

Protein Purification Protocols Methods in Molecular Biology, Vol 244

The optimal approach often must be determined empirically. The best protein purification protocol depends not only on the protein being purified but also on many other factors such as the cell used to express the recombinant protein e. Escherichia coli remains the first choice of many researchers for producing recombinant proteins due to ease of use, rapid cell growth and low cost of culturing. Proteins expressed in E.

Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications Geisse et al. However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge for their purification. As a result, attaining satisfactory yield and purity depends on highly selective and efficient capture of these proteins from the crude cell lysates.

To simplify purification, affinity purification tags can be fused to a recombinant protein of interest Nilsson et al. Common fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. The biochemical features of different tags influence the stability, solubility and expression of proteins to which they are attached Stevens et al. Using expression vectors that include a fusion tag facilitates recombinant protein purification.

A major objective in proteomics is the elucidation of protein function and organization of the complex networks that are responsible for key cellular processes. Analysis of protein:protein interactions can provide valuable insight into the cell signaling cascades involved in these processes, and analysis of protein:nucleic acid interactions often reveals important information about biological processes such as mRNA regulation, chromosomal remodeling and transcription.

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This regulation is required for cell viability, differentiation and growth Mankan et al. Analysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function.

Protein Expression & Purification

This immobilization must not interfere with the binding capacity and can be achieved through the use of affinity tags. Immobilization of proteins on chips is a popular approach to analyze protein:DNA and protein:protein interactions and identify components of protein complexes Hall et al. Functional protein microarrays normally contain full-length functional proteins or protein domains bound to a solid surface.

Fluorescently labeled DNA is used to probe the array and identify proteins that bind to the specific probe. Protein microarrays provide a method for high-throughput identification of protein:DNA interactions. Immobilized proteins also can be used in protein pull-down assays to isolate protein binding partners in vivo mammalian cells or in vitro. Other downstream applications such as mass spectrometry do not require protein immobilization to identify protein partners and individual components of protein complexes. One method for isolating or immobilizing a specific protein is the use of affinity tags.

Many different affinity tags have been developed Terpe, Fusion tags are polypeptides, small proteins or enzymes added to the N- or C-terminus of a recombinant protein. The most commonly used tag is the polyhistidine tag Yip et al. Protein purification using the polyhistidine tag relies on the affinity of histidine residues for immobilized metal such as nickel Yip et al. This affinity interaction is believed to be a result of coordination of a nitrogen on the imidazole moiety of polyhistidine with a vacant coordination site on the metal.

The metal is immobilized to a support through complex formation with a chelate that is covalently attached to the support. Polyhistidine tags offer several advantages for protein purification. The small size of the polyhistidine tag renders it less immunogenic than other larger tags. Therefore, the tag usually does not need to be removed for downstream applications following purification.


A polyhistidine tag may be placed on either the N- or C-terminus of the protein of interest. Finally, the interaction of the polyhistidine tag with the metal does not depend on the tertiary structure of the tag, making it possible to purify otherwise insoluble proteins using denaturing conditions. Glutathione-S-transferases are a family of multifunctional cytosolic proteins that are present in eukaryotic organisms Mannervik and Danielson, ; Armstrong, The 26kDa GST affinity tag enhances the solubility of many eukaryotic proteins expressed in bacteria.

Protein fusion tags are used to aid expression of suitable levels of soluble protein as well as purification. The synthetic linker can be attached to a variety of entities such as fluorescent dyes and solid supports to allow labeling of fusion proteins in cell lysates for expression screening and capture of fusion proteins on a purification resin.

HaloTag is a powerful technology with applications for protein purification, protein localization, trafficking and turnover as well as protein interactions and super-resolution microscopy. The combination of covalent capture and rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. FIgure 1.

Protein Purification and Analysis

This interaction is highly specific and irreversible. There is a growing need for high-throughput protein purification methods. Magnetic resins enable affinity-tagged protein purification without the need for multiple centrifugation steps and sequential transfer of samples to multiple tubes. There are several criteria that define a good protein purification resin: minimal nonspecific protein binding, high binding capacity for the fusion protein and efficient recovery of the fusion protein.

The magnetic nature of the binding particles allows purification from crude lysates to be performed in a single tube. In addition, the system can be used with automated liquid-handling platforms for high-throughput applications. Polyhistidine-tagged protein can be purified on a small scale using less than 1ml of culture or on a large scale using more than 1 liter of culture.

Polyhistidine-tagged proteins can be purified under native or denaturing 2—8M urea or guanidine-HCl conditions. The presence of serum in mammalian and insect cell culture medium does not interfere with purification. Purification using Denaturing Conditions. Proteins expressed in bacterial cells may be present in insoluble inclusion bodies. Alternatively, if quantity of tissue is not a problem, the humble laboratory rat may suffice. Once a protocol for purifying the protein from substitute sources has been worked out, it will be much easier to develop one using human material—the identical procedure may work satisfactorily.

Proteins differ to a fairly small extent between species that have diverged within about million years, a time frame that groups together most higher mammals. Thus the behavior of proteins derived from different animals with respect to the various fractionation procedures is likely to be similar, and a protocol worked out for pig tissues is likely to need only minor adjustments for application to human tissues.

The methods available for protein purification range from simple precipitation procedures used since the nineteenth century to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. Methods can be classified in several alternative ways—perhaps one of the best is based on the properties of the proteins that are being exploited. Thus the methods can be divided into four distinct but interrelated groups depending on protein characteristics: surface features, size and shape, net charge, and bioproperties.

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