| Before beginning, you will likely be faced with several decisions. For example, do you want to use an in vivo or in vitro expression system? Most of the in vivo systems are used for bacterial expression, but they can also be used in mammalian, insect, or yeast cells. Bacteria are widely used because they generate higher yields of protein. Expression in eukaryotic cells yields less, but allows mammalian proteins to be post-translationally modified in a fashion closer to native conditions. You will also need to choose whether to tag the amino or carboxyl terminus (or both in some cases) of your fusion-protein-to-be. You may need to choose the promoter type for expression, and whether your protein is secreted or cytoplasmic. You also need to consider scale—in other words, how much protein you need for your experiments. For protein expression on a smaller scale, in vitro expression (discussed later) may be ideal.
If this seems like a lot to deal with, do not despair—many of these considerations will be made for you depending on which kit you choose. For example, many kits don’t offer a range of promoter types, so it’s one less thing to worry about if you don’t have a promoter preference. Probably the most important thing to consider is the type of tag that best suits your purposes. For example, you may choose a kit based on tag size, tag solubility, tag toxicity to the expressing organism, ease of purification, the possibility of double-tagging or protein engineering, tag removal, or the possibility of in vitro protein expression. These criteria will be discussed in the context of some current kit offerings.
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| Tags come in a range of sizes, from a few amino acids to tens of kiloDaltons. Depending on your plans for your purified protein, the size of your tag may be important, especially if the tag is near a functional region after folding. The well-studied GST (glutathione-S-transferase) tag is one of the most widely used tags, but at 26 kDa, it is also relatively large. A disadvantage of the GST tag is that purification using glutathione must be performed under non-denaturing conditions to ensure the functionality of the GST-glutathione interaction. Using denaturing conditions may be necessary if your protein is difficult to get into solution, says Doug Hayworth, Senior Technical Assistance Representative at Pierce, citing an example of proteins that are packaged into inclusion bodies when expressed in bacteria.
An expression and purification system that may help to solubilize your protein is offered by New England BioLabs. Their pMAL System is designed to tag and express proteins tagged with maltose-binding protein (MBP). Your protein is fused to the C-terminus of MBP, then bound to an amylose column and eluted with maltose. At 40 kDa, the MBP is even larger than a GST tag; nonetheless, using a MBP tag can be advantageous because it may make your protein more soluble. “An additional advantage of the pMAL System,” says James Ellard, Director of Marketing Communications at New England BioLabs, “is that it also includes a vector that can export the protein to the periplasmic space, which can aid in the folding of secreted proteins, especially those with disulfide bonds, and can help cells avoid the effects of toxic proteins.”
If you require a tag that can withstand denaturing conditions, the commonly used His tag may be a good choice. Comprised of 6-10 histidines, it also has the advantage of being smaller than the GST or MBP tags. A disadvantage of the traditional His tag is that during affinity purification, other proteins (especially those containing several histidines) may bind nonspecifically to the nickel matrix typically used to selectively immobilize your tagged protein—resulting in contamination of your protein sample. Qiagen aims to remedy this problem with the purification products of their QIAexpress® System, which allows you the flexibility of expressing 6xHis-tagged protein in E. coli, insect, or mammalian cells. The kits include expression vectors, host strains of E. coli, matrices for chromatography, and antibodies specific to the affinity tags. The 6xHis tag “is the most widely used affinity tag and has therefore become an industry standard,” says Anke Cassing, Associate Director of Corporate Strategy at Qiagen. With Qiagen’s Ni-NTA (nickel-nitrilotriacetic acid) chromatography matrix for the purification of His-tagged proteins, Cassing says, “more than 95% purity can be achieved in a one-step procedure.” If removal of the His tag from the protein is desired, it can be removed enzymatically using Qiagen’s TAGZyme® System.
While His tags are widely used, they are not always appropriate—in the purification of metal-binding proteins, for example, or if the buffer contains a metal chelator such as EDTA. The QIAexpress® System also offers an alternative, the Strep tag. Only 8 amino acids long, the Strep tag binds with high affinity and specificity to Qiagen’s engineered streptavidin matrix, Strep-Tactin®. For those who can use a His tag and want an additional measure of purification, the QIAexpress System allows you to use both the His and Strep tags. Double-tagging, or putting a different tag on each end of your protein, can yield greater purity by enabling two rounds of affinity purification. “Proteins carrying a 6xHis tag and Strep-tag can be purified by sequential affinity purification on Ni-NTA and Strep-Tactin matrices to deliver ultrapure recombinant proteins expressed in E. coli, insect, or mammalian cells,” explains Cassing.
If you need a small tag, or one that allows purification with the |
So you have a gene that you want to express…and you’d like to generate a sample of purified protein quickly and easily? It sounds like you’re in the market for an expression and affinity purification kit. For someone with little background in this area, the possibilities can bewilder. Even for the experienced gene jock, new and improved products may hit the market before you take notice. The kits discussed below should help to put you on the right track toward successful protein expression. You’ll likely want to buy a kit that allows you to place an affinity tag on your protein, because a tag will allow affinity purification of your protein and can be helpful in other applications, such as detection and subcellular localization. An important place to start is choosing the type of tag.
