Bench philosophy: Inteins

Protein Splicing Specialists
by Steven Buckingham, Labtimes 04/2015

A battery of interesting new inteins has burst onto the scene, providing a vital link between genetic engineering and classical protein chemistry.

What are inteins? Inteins are proteins that are capable of splicing out a part of themselves, just like RNA splicing. Indeed, so close is the analogy with RNA splicing that the very name of "intein" is borrowed from the RNA world of introns and exons. You'll see why in a minute, once I have told you how inteins work.

Rope makers use a marlin spike or a fid to splice ship ropes. Life scientists may apply inteins for splicing and modifying protein strands.

Three-part structure

Structurally, inteins have three parts − a central intein domain flanked on either side by an extein (inteins/introns; exteins/exons − get the point?). The regions where the intein and extein domains meet are capable of cis-splicing: in a series of exquisitely coordinated steps, the intein/extein boundary is cut and the two resulting exteins get instantly ligated back together. Result: one protein made of the two fused exteins plus the now-isolated intein domain.

Intein splicing is found naturally in both prokaryotes and eukaryotes. Most of them contain a homing DNA endonuclease domain in the middle of the intein domain, which participates in an outlandish "selfish gene" activity, where the intein propagates itself by inserting sequence back into the genome. But you don't need this endonuclease domain for it to work − in fact, some natural inteins don't have them in the first place.

Intein splicing is found in many bacteria and fungi but has not yet been found natively in animals. It is much rarer than RNA splicing, although about 500 inteins have been found, so far. There is a database of inteins kept at InBase ( Scientists may argue about exactly what part it plays in nature but for most of us only one thing matters: its increasingly important place in the bench biologist's toolbox.

Controllable and self-splicing

After all, just think of the possibilities. Start thinking like a protein chemist and ask yourself, what you could do with a protein that actually splices itself. And here's another big plus: you can even control when the intein splices itself, by adding a signalling reagent or just changing the temperature. What a gift − a controllable, self-splicing protein! Let the ideas flow...

Early applications

That's what developers in New England Biolabs did back in the late 1990s, when they realised the potential of inteins for protein purification. Purifying a protein usually relies at some point on high affinity binding, which is a bit of a two-edged sword. When it comes to getting your synthesised protein to stick to a surface so you can harvest it, affinity is your friend. But it turns out to be a bit of a bind when the time comes to strip the protein off again.

This is where NEB's modified inteins came in. The system works by using an intein to bridge the expressed protein and its affinity tag. The tag-intein-protein complex sticks to your surface and gets immobilised. Then you change the pH to activate the intein and the intein domain releases the protein. And this can happen in as quickly as 30 minutes (at least, using more modern inteins).

But that was some 20 years ago. What has been happening since? There have been two parallel developments that have led to an increasing set of applications of inteins. The first is that new inteins have been found in a variety of species, providing an increasing set of starting points for engineering new intein applications. For one, inteins sensitive to different environmental stimuli have been found, increasing the set of potential triggers you can use to spark off the splicing reaction.

Splicing in trans

A particularly important development in this area is the discovery and development of trans-splicing inteins. This means you can put each of the two complementary intein domains onto each of two exteins, so that splicing only occurs when the two constructs come together.

The second development has been the steady growth in new applications of the expanding intein toolkit, as researchers have become aware of the potential of protein splicing. For one, inteins are a convenient way of modifying the C-terminus of a protein. One complementary strand of a trans-splicing intein is fused to the target protein and the other is fused to the chemically-modified C-terminus fragment. Inteins also provide another way of achieving conditional expression of transgenic proteins. In all these cases, the idea is to place the activation of the inteins under some form of trigger, whose activity results in the splicing of two exteins into some sort of active protein.

Genetic marker

Inteins have a lot of potential as a new kind of genetic marker. You insert a genetic marker into an intein domain and the good news is yes, you can do that without breaking the intein's ability to splice. Eric Muller and his co-workers at the University of Washington, USA, did this using selectable markers, such as aminoglycoside phosphotransferase (Ramsden et al., 2011 BMC Biotechnol 11:71). For the exteins, you use two halves of a label − GFP will do the trick. When the genetic marker is activated, the intein is spliced and fully functional GFP is released.

Inteins are useful for tracking protein-protein interactions. All you do is attach the two complementary, trans-splicing intein strands to each of the two proteins you are interested in, along with half of a reporter protein. When the proteins start sidling up to each other, the intein strands pair up and start splicing, resulting in a full-length, active reporter.

Post-translational modification

One particular reason for the surge in interest in inteins is because the technique allows you to do post-translational modifications of proteins in a controlled and specific way. Want to phosphorylate a specific protein on the cell surface? You can do it with inteins. First, you express a protein on a cell surface lacking the part you want to phosphorylate. Stick on a trans-splicing intein strand facing away from the membrane. Next, stick the complementary intein onto a protein fragment that you have chemically modified − phosphorylated in this case. Perfuse the cells with this and when the intein strands join up (you can speed this up by adding a receptor/ligand pair to the intein strands as a sort of match-maker), the intein domain will glue the protein together and discretely leave.

Inteins also come in handy when you want to express a protein complex with a single open reading frame. Antibodies, for instance. You can express the light chain as one extein and the heavy chain as the other, linked by an intein. Activate the intein and the two molecules get processed together.

Stealth mode

You can also get a helping hand from inteins when you are having problems transforming a large gene. Large constructs can be hard to insert into cells, because of the intrinsic limitations of vectors. By attaching complementary intein strands to each half-gene, you can stealthily insert the gene in instalments, leaving the inteins to gather up the pieces and patch them back together.

Inteins can open up possibilities for the production of highly toxic proteins. Synthesising a toxic protein has its obvious challenges. But inteins can often provide a way forward. One way to do this is to get the cells to produce the toxic protein in two (non-toxic) halves, using trans-splicing intein strands on each half to direct the ­reassembly, under the control of a trigger. Alternatively, express the toxic protein interrupted by an intein in such a way as to render the protein innocuous. Again, final assembly of the toxic protein is induced by triggering intein splicing.

Improved safety

Inteins can be used to make safer transgenic organisms. Public confidence in genetically modified organisms is − perhaps rightly − undermined by the fear of transgenic material leaking out into the environment through horizontal gene transfer. This is less likely to happen when the gene is split into two and inserted into remote parts of the genome. A complementary intein strand on each half will ensure that the two halves get stitched up together, in place.

In addition to helping out with protein manufacture, inteins have proved their worth as reporters of basic cellular processes. The redox state of cell compartments, for example, is an important factor in cell function but can be hard to record. Usefully, some inteins available are inactivated by the presence of a dicysteine bond. Changes in redox can result in breaking the dicysteine bond and consequently activating the intein, resulting in the fusion of the exteins. If you are using an intein to trace cellular processes, what sort of read-outs are available? The most obvious approach is for the exteins to encode two halves of a reporter. Intein splicing yields a full-length active reporter, such as a fluorescent protein, and the cells light up. But you can do it the other way around, too: there are inteins that only do half the job − just cutting at one end but not re-ligating. Here, reporting can be done using FRET (Fluorescent Resonance Energy Transfer). While the intein is inactive, it clings on to both FRET partners (CFP or YFP, for example) but when it is activated, it cuts at one of the intein-extein boundaries, releasing its exteins. This is monitored as a decrease in the FRET signal.

Self-processing maize

Inteins may even turn up in our food. A group from the Massachusetts-based animal nutrition company, Agrivida, used inteins to produce self-processing maize (Shen et al., 2012 Nat. Biotechnol. 30, 1131-36). They transfected the maize with the gene (xylanase from Dictyoglomus thermophilum) that converts starch to sugar. If you do that in the conventional way, though, you destroy the seeds − they shrivel up and are no good for planting.

But Shen's group split the xylanase gene into two parts and used an intein to allow the plants' cellular machinery to produce the xylanase in place. The team used directed evolution to come up with a version of intein that was activated specifically at high temperature. So, you can grow and propagate the maize just like normal but when the time comes you want it to process itself, store it in the airing cupboard.

Bridging the gap

Inteins are an exciting tool because they sit at the interface between genetic engineering and classical biochemistry. They open up a range of biological questions that are unanswerable using genetic techniques alone. By bridging molecular genetics with protein chemistry, intein technology brings to the bench new ways of doing old things (genetic markers, conditional expression) and new ways of doing new things.

Last Changed: 08.07.2015