Proteins can be purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation, electrophoresis, and chromatography. This process usually begins with cell conditional media (for secreted proteins), or cell lysates in which a cell's membrane is disrupted and its internal contents released into a crude extract (for intracellular proteins). The resulting starting materials can be purified by fractionation of the extracellular or intracellular components. Various types of chromatography are used to isolate the protein of interest based on its physical and chemical properties, such as size, net charge, hydrophobicity and affinity binding group. Genetic engineering is often used to add tags or fusions to help purify specific proteins from complex mixtures. For example, His-tag and Fc-fusion are the most commonly used techniques in protein preparation (see the list and discussion in the previous topic).

The objective of a protein purification process is to yield the maximal amount of functional protein with minimal contaminants. The purification procedure should be optimized to complete the process using the minimum number of steps in the least amount of time and ensure the highest yield of functional protein. Another important consideration is the downstream application of the purified product. Both the quantity and purity must be sufficient for experimental analysis. In addition the properly-folded and functional protein is normally required for downstream studies. During purification and subsequent storage, many events can occur that affect protein quality and stability, including protein unfolding, aggregation, degradation, and loss of activity. Careful planning to purify protein as quickly as possible under the optimal stabilizing conditions will maximize the chance of a successful purification process.

Chromatography in Protein Purification

Affinity Chromatography

Affinity chromatography relies on the specific and reversible binding of a protein to a matrix-immobilized ligand. The ligand can either bind directly to the protein of interest or to a tag that is genetically engineered to attach to the protein. Proteins can also be affinity purified in a non-selective manner when the immobilized ligand binds to a group of proteins with similar binding behaviors. Affinity chromatography is often the most robust purification procedure and typically used in early stages of a purification process. Depending on the downstream application, affinity purification might be the only chromatographic step required to achieve adequate purity. Two most commonly affinity chromatographic methods in recombinant protein purification are immobilized metal (Nickel) and protein A/G affinity chromatography.

Immobilized Metal Affinity Chromatography

Six or more tandem histidines form a metal-binding structure. Thereby proteins tagged with poly-histidines (His-tag) can be purified by affinity to nickel or cobalt using Immobilized Metal Affinity Chromatography (IMAC) columns. His-tag has become the most commonly used method for protein purification and preparation in almost all expression systems, including the mammalian systems. IMAC is the widely employed because it has the particular advantage of specific binding and eluting conditions that are less damaging to the protein than other methods. For example, a nickel-nitrilotriacetic acid (Ni2+-NTA) chelate can be covalently attached to a cross-linked agarose matrix for the selective purification of His-tagged recombinant proteins (see the diagram below).

His-tag can coordinate to the Ni2+ ion by replacing the bound water molecules (indicated by two red arrows). Imidazole can then be used to elute the protein, through its ability to coordinate with the Ni2+ ion and displace the bound protein.

Antibody Affinity Chromatography

Antibodies contain several regions of interest: Fc (fragment, constant) that is highly homologous for all antibodies of the same class, and Fab (fragment, antigen binding) that makes direct contact with the specific antigen. Specific classes of antibodies can be non-selectively purified through affinity chromatography in which the ligand, Protein A, G, or L, is conjugated to a cross-linked matrix. For example, protein A or G affinity matrix allows the rapid and efficient purification of IgGs and derived Fc-fusion proteins. Typically one-step of protein A or G affinity chromatography can yield products with more than 90% purity. Protein A, G, or L can also be used to purify specific proteins. In this case the antibody will serve as an intermediate ligand providing selectivity for its antigen (see the diagram and discussion below).

Antibodies are often purified based on the highly specific interaction that occurs between an antibody and its antigen or immunogen. For example, a peptide containing the antigen can be coupled to a matrix for specific binding of the antibody. Lowering the pH of the elution buffer disrupts the antibody and peptide interaction to release the bound antibody. This method is routinely used in the isolation of specific antibodies from polyclonal anti-sera. Using a similar mode of binding (e.g., using an immobilized antibody on protein A or G), the antigen-binding (Fab) region of the antibody is still available for binding to its specific antigen. Thus, a specific target protein or antigen can be purified based on the specific interaction with the coupled antibody and eluted with competing antigen, such as a peptide containing antibody-recognizing epitope.

In many cases, purity is often not enough using just one or a few steps of affinity purifications. It is important to add additional purification steps, such as ion exchange and size exclusion chromatography.

Ion Exchange Chromatography

Ion exchange chromatography (IEX) separates proteins based on their net surface charge, through electrostatic interactions that occur between proteins and a charged matrix. Two types of IEX exist:

Ion exchange chromatography is commonly used as an intermediate step in a protein purification process; however, it can yield high resolution for some proteins when used earlier or later during the purification. All proteins exhibit a net charge that depends on the amino acid composition of the protein and the pH of buffer used.

Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) separates proteins based on their hydrophobicity, and is often used as an intermediate step in a purification process. Proteins are bound to a matrix in a high ionic strength buffer and HIC is typically performed immediately after ion exchange chromatography with no buffer exchange or dilution required. HIC is also commonly performed after an ammonium sulfate precipitation, a procedure that can be used to quickly concentrate or remove proteins by precipitating some, but not all, proteins with salt. HIC can also be applied in early steps of a purification process or as a final step in the removal of trace impurities from the protein of interest.

Size Exclusion Chromatography

Size exclusion chromatography separates proteins by their hydrodynamic radius, a characteristic determined by both the size and shape of the protein. Unlike the other chromatographic procedures described previously, proteins do not bind to a cross-linked matrix in SEC. Instead, proteins are separated by the speed at which they migrate through the matrix. SEC is most often used in the final steps of a purification process due to its ability to differentiate between different forms of a protein. For example, oligomeric, unfolded, and degraded forms can all be separated from the native protein, while simultaneously exchanging the buffer system. Therefore SEC has also been used as a faster and reliable method for buffer exchange.

G&P Biosciences Protein Production Capability

G&P Biosciences benefits greatly from a number of technical advantages, providing the best chance of expressing a protein in soluble, active form with high yield. We have experience and expertise in all stages of protein expression from codon optimization, vector/promoter choice, tag/fusion use, culture medium, additives, feedings, host cell engineering, stable cell line creation to protein purification and formulation (see a typical workflow below). We develop unique, high throughput, mammalian cell expression systems, allowing exploiting every technique to improve the productivity for recombinant protein expression. With innovative solutions for varying conditions & parameters, we can quickly deliver recombinant proteins with the highest qualities possible to our clients in a short turnaround time. 

We offers custom recombinant protein expression and purification using mammalian cell expression systems (in particular HEK293 and CHO) for small or large quantities (top to grams) of protein production. Our unique mammalian cell expression systems that are based on our high-throughput gene expression technologies, exhibit significantly improved success rates and production yields.  Our custom-made recombinant proteins are expressed in mammalian cells growing under animal component-free, antibiotic-free condition, purified to the highest homogeneity possible, and formulated into the carrier-free or customer-specified buffer suitable for downstream in vitro & in vivo assays (visit our "Protein Products" and "Protein Services" pages to learn more and order).

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