Protein Purification Methods

Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. 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. The optimal approach often must be determined empirically.

Protein Purification

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.g., prokaryotic versus eukaryotic cells). 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. coli can be purified in relatively high quantities, but these proteins, especially eukaryotic proteins, may not exhibit proper protein activity or folding. Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications (Geisse et al. 1996). 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. 1997). 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. 2001). Using expression vectors that include a fusion tag facilitates recombinant protein purification.

Isolation of Protein Complexes

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. For example, transcription factors play an important role in regulating transcription by binding to specific recognition sites on the chromosome, often at a gene’s promoter, and interacting with other proteins in the nucleus. This regulation is required for cell viability, differentiation and growth (Mankan et al. 2009; Gosh et al. 1998).

Analysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. 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. 2004; Hall et al. 2007; Hudson and Snyder, 2006). 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.

The lack of an endogenous equivalent of the HaloTag® protein in mammalian cells minimizes the chances of detecting false positives or nonspecific interactions. 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. In addition, the highly stable HaloTag® protein:ligand interaction permits boiling the protein complex in SDS sample buffer prior to SDS-PAGE analysis.

Purification of Polyhistidine-Tagged Proteins

Rapid Purification of Polyhistidine-Tagged Proteins Using Magnetic Resins

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 MagneHis™ Protein Purification System meets these criteria, enabling purification of proteins with a broad range of molecular weights and different expression levels. 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.

MagneHis™ Protein Purification System

The MagneHis™ Protein Purification System uses paramagnetic precharged nickel particles (MagneHis™ Ni-Particles) to isolate polyhistidine-tagged protein directly from a crude cell lysate. Figure 2 shows a schematic diagram of the MagneHis™ Protein Purification System protocol. 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. Samples can be processed in a high-throughput manner using a robotic platform such as the Beckman Coulter Biomek® FX or Tecan Freedom EVO® instrument. 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. For more information and a detailed protocol, see Technical Manual #TM060 and the MagneHis™ Protein Purification System Automated Protocol #EP011.

Protocol: MagneHis™ Purification of Proteins Expressed in Bacterial Cells

Materials Required:

• MagneHis™ Protein Purification System and protocol
• 37°C incubator for flasks or tubes
• shaker
• magnetic separation stand
• 1M imidazole solution (pH 8.0; for purification from insect or mammalian cells or culture medium)
• additional binding/wash buffer (may be required if processing numerous insect cell, mammalian cell or culture medium samples)
• solid NaCl (for purification from insect or mammalian cells or culture medium)

Purification using Denaturing Conditions. Proteins expressed in bacterial cells may be present in insoluble inclusion bodies. To determine if your protein is located in an inclusion body, perform the lysis step using FastBreak™ Cell Lysis Reagent, 10X, as described in Technical Manual #TM060. Pellet cellular debris by centrifugation, and check the supernatant and pellet for the polyhistidine-tagged protein by gel analysis.

Efficient purification of insoluble proteins requires denaturing conditions. Since the interaction of polyhistidine-tagged fusion proteins and MagneHis™ Ni-Particles does not depend on tertiary structure, fusion proteins can be captured and purified using denaturing conditions by adding a strong denaturant such as 2–8M guanidine hydrochloride or urea to the cells. Denaturing conditions must be used throughout the procedure so that the proteins do not aggregate. We recommend preparing denaturing buffers by adding solid guanidine-HCl or urea directly to the MagneHis™ Binding/Wash and Elution Buffers. For more information, see Technical Manual #TM060.

Note: Do not combine FastBreak™ Cell Lysis Reagent and denaturants. Cells can be lysed directly using denaturants such as urea or guanidine-HCl.

Purification from Insect and Mammalian Cells. Process cells at a cell density of 2 × 10⁶ cells/ml of culture. Adherent cells may be removed from the tissue culture vessel by scraping and resuspending in culture medium to this density. Cells may be processed in culture medium containing up to 10% serum. Processing more than the indicated number of cells per milliliter of sample may result in reduced protein yield and increased nonspecific binding. For proteins that are secreted into the cell culture medium, remove any cells from the medium prior to purification. For more information, see Technical Manual #TM060.

HaloTag® Protein Purification from Mammalian Cells

Cultured mammalian cells offer an environment well suited for producing properly folded and functional mammalian proteins with appropriate post-translational modifications. However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge. As a result, attaining satisfactory yield and purity depends on selective and efficient capture of these proteins from the crude cell lysate. The equilibrium-based binding of most affinity tag protein purification methods means that the protein is constantly being exchanged between the bound (to the resin) and unbound state. This equilibrium depends on the protein concentration and binding affinity of the tag. As a result, binding efficiency may be reduced at low expression levels, leading to low recovery of the fusion protein.

The HaloTag® Mammalian Protein Purification Systems (Cat.# G6795 and Cat.# G6790) use the HaloTag® protein tag, which can be genetically fused to any protein and transiently or stably expressed in mammalian cells. Following cell lysis, the HaloTag® fusion protein is covalently captured on the HaloLink™ Resin, and nonspecific proteins are washed away. The protein of interest is released by a specific proteolytic cleavage at an optimized TEV recognition site contained within the amino acid linker sequence that connects the HaloTag® protein tag and protein of interest. To eliminate the need for a secondary step to remove the protease, TEV protease fused to HaloTag® (HaloTEV Protease; Cat.# G6601) can be used to cleave the HaloTag® fusion protein and then covalently captured on the HaloLink™ Resin, resulting in a streamlined purification process. This straightforward purification uses a single, mild physiological buffer throughout the entire process with no need for buffer exchange.

HaloTag® Protein Purification from E. coli

The HaloTag® Protein Purification System (Cat.# G6280) allows covalent, efficient and specific capture of proteins expressed in E. coli as N-terminal HaloTag® fusion proteins. Many of the same characteristics that make the HaloTag® protein well suited for purifying proteins from mammalian cells also make it a good choice for purifying proteins from E. coli cells. HaloTag® fusion proteins can be expressed in E. coli using a number of expression vectors specifically designed for E. coli including the pFN18A HaloTag® T7 Flexi® Vector (Cat.# G2751) and pFN18K HaloTag® T7 Flexi® Vector (Cat.# G2681) as well as non-Flexi® vectors, which are available with dual tags of HaloTag® protein and polyhistidine. These non-Flexi® vectors, pH6HTN His6HaloTag® T7 Vector (Cat.# G7971) and pH6HTC His6HaloTag® T7 Vector (Cat.# G8031), allow traditional cloning using the multiple cloning site. These dual-tagged vectors enable purification of HaloTag® fusion proteins that still retain the covalent coupling ability of the HaloTag® protein. With the HaloTag® Protein Purification System, it is easy to perform in-gel detection and quantification of protein expression levels using fluorescent HaloTag® Ligands.

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