Wayne K. Way, Ph.D., Head of Protein Preparation and Reagents, MilliporeSigma, Bellefonte, PA,
George C. Yeh, Ph.D., Product Manager, Protein Purification, MilliporeSigma, St. Louis, MO,
Many biopharmaceuticals are recombinant proteins obtained by biotechnological processes. In various steps of the pharmaceutical value chain, from drug discovery, to development and manufacturing, to bioprocessing, these proteins must be extracted from their complex biological sources, typically microorganisms or genetically modified cells. The target proteins are subsequently purified and their presence confirmed by assay.
However, proteins are more challenging to purify than macromolecules like DNA or RNA, which can be targeted with complementary oligonucleotides. The great diversity of natural protein sequences and the lack of the native complementary structures that DNA or RNA possess means that scientists must use a different route to purification.
Affinity tag considerations
A key approach in recombinant protein purification is to use affinity tags, also known as fusion tags or epitope tags. These are additional peptide sequences, or in some cases even full-length protein sequences, that have been engineered into the recombinant protein at the outset to provide a way of capturing the expressed target protein by affinity chromatography. Such tags can be as small as six amino acids, like the 6-histidine-tag (His-Tag™), or eight amino acids, like the FLAG® tag, known also as DYKDDDDK. Conversely, an example of a large molecular weight protein tags is glutathione-S-transferase (GST), sized about 27 kDa.
Many factors influence the tagging strategy, including the size and the location of the tag. Larger protein tags are more likely to interfere with a protein’s folding and function, hence small, peptide-sized tags like FLAG®, the His-Tag™, or the hemagglutinin (HA) tag, have become very popular. The tag is usually placed at either the N-terminus or the C-terminus of the protein, although in principle, it can be engineered anywhere in a given protein. Large protein tags, on the other hand, are often easier to detect because they are sterically more accessible to the antibodies that are supposed to bind them.
Another consideration is protein solubility, especially when the protein of interest is expressed at very high levels and its solubility thus becomes an important concern. For this reason hydrophilic tags, which enhance the solubility of the target protein upon expression at high levels, tend to be preferred. The FLAG® tag and the His-Tag™ are prime examples of such hydrophilic tags, because of their high degree of charge.
The matching chromatography resin
Some tags allow the recombinant protein to be purified by inexpensive methods, for example poly-histidine by immobilized metal affinity chromatography, but the purity levels achieved are often disappointing. Chromatography resins with bound antibodies that are specific for the protein tag of interest generally achieve higher levels of protein purity. There is a cost, though, as antibody resins are expensive to produce and difficult to reuse. However, the high purity from using antibody resins often improves cost-effectiveness in the long run, as secondary purification steps can be avoided. This is of particular interest in high-throughput screening, or where proteins are expressed at low levels, for example while expression conditions are being optimized.
There are several formats for chromatography media to capture recombinant proteins. The most common are cross-linked agarose conjugates of antibodies to specific protein tags, which can be used in batch column or multiwell plate format. Of late, magnetic agarose beads (“magnetic beads”), which contain a magnetic core surrounded by cross-linked agarose, are increasingly being used to purify recombinant proteins because of their time-saving qualities and speed when used with a suitable magnet to capture the beads.
If the biotherapeutic is an antibody there is a more elegant purification strategy available than tagging. Here, the long-standing procedures center around using Protein A, Protein G, and Protein L as affinity ligands to capture the therapeutic antibodies. These proteins have an inherent native affinity for immunoglobulins, so there is no need to use specific antibodies for capture.
Removing the tag
In some instances, the protein tag must be removed after purification of the target protein. Various proteases exist to remove such tags. The enzymes are specific to given sequences where the tag is attached, allowing them to selectively cleave the tag.
While there are scientists that come up with novel tags for niche uses, most are generally reluctant to give up the well-established tags they have been using. This is because a new protein tag means having to use new cloning and expression vectors to express the protein with the tag, along with a new suitable chromatography resin to capture the protein with its novel tag. However, if a combination of tag and chromatography resin is well-tried and promises greater purity, it should be worth considering.
See our extensive range of products for protein and antibody purification, which includes magnetic beads, agarose gels, enzymes, peptides, antibodies, vectors, coated plates, FLAG® purification tools and more.
Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special offers
from American Pharmaceutical Review – all delivered right to your inbox! Sign up now!