U of Chicago protein engineer Shohei Koide knows there’s biology beyond the genome. Adding a methyl group onto a histone here, or an acetyl group onto one there, can change the way genes are expressed. There’s gotta be a reliable way of finding those postranslational mods in the first place, though, right? That’s why he jumped into the epigenomics game, ending up with an NIH Roadmap grant for Technology Development. “I find pleasure in making useful tools that would enable other people to do something they couldn’t do before.”
Koide’s lab works on affinity clamps, a family of capture reagents, to use for ChIP, mass spec, peptide arrays, and the like. “The term ‘affinity clamp’ comes from the fact that our design uses basically 2 domains to literally clamp over very small peptides.”
They’re composed of two molecules with weak affinity for a peptide, fused together via a linker, and refined by in vitro directed evolution methods. “We can fine-tune experimental conditions to isolate the type of molecules with the right characteristics. Imagine grabbing a snake — affinity clamps are like a glove, with a thumb and fingers — from both sides.
“When you think about capture reagents you need to think about 2 things: affinity and specificity. Our results so far suggest that affinity clamping is a very good way to achieve very high levels of both.”
Koide and his colleagues last year filed a patent on what he calls a “conceptual breakthrough in protein engineering”.
So what’s in it for epigenetics research?
“We are particularly interested in making this type of technology for capturing floppy peptides — a good example is histone tails carrying post-translational modifications. These are not nicely folded proteins, and they’re difficult to capture.”
The near-term goal – with respect to chromatin – is two-fold. The first is to produce ‘histone clamps’ “to discriminate methylation at different sites — H3K7 or H3K27, for example. The second is to discriminate levels of methylation: mono-, di-, and tri-. … Using in vitro directed evolution, we can set up experiments in such a way that only those molecules that can, for example, discriminate tri-methyl from di-methyl can survive our selection pressure.
Affinity Clamps vs. Antibodies
Why not just use antibodies?
“Antibodies typically have a very flat surface, and are not particularly good at capturing this type of molecule.”
Besides, there are very few good monoclonals to this type of posttranslational mod, and there’s so much variability in polyclonals. “We’re interested in eliminating sample-to-sample differences and getting consistency and reproducibility of experiments.”
“Our current focus is to make high quality capture reagents — highly validated and stable reagents with which you can do the same experiment now or in ten years and nothing changes.
“These proteins are produced in E. coli. We can make a huge amount of protein easily — unlike the polyclonal antibodies most people are currently using in ChIP experiments, our reagents are recombinant and by definition monoclonals. We can always scale up what we produce without changing properties.
Are affinity clamps just better antibodies?
To some extent they’re complementary – at least for now. In fact, Koide’s lab works on both synthetic antibodies and affinity clamps.
“Antibodies already have a good infrastructure – an already-established pipeline. … They’re generic binding reagents, which is both strength and a weakness. With an affinity clamp if we were going to switch from methylation to acetylation, we would have to build a whole system.”
Essentially, anything that can be done with antibodies can theoretically be done with affinity clamps.
On the other hand, “a real transformative effect could come from new applications that we cannot do with antibodies. For example, because affinity clamps are genetically encoded, we could express them in cells. In principle you could use them in live cell imaging. … I think it would be quite exciting to be able to see where the histone modifications are located within live cells and how they change over time. Where different modifications are localized, if they are.
Timelines for Affinity Clamps
“Our goal is to get hopefully a half-dozen to a dozen reagents by the end of this initial phase of the funding – that means next summer. … Our current focus is to make high quality capture reagents to most likely studied marks.”
Affinity clamps will be validated in collaboration with several epigenetics labs. Expression vectors will probably be deposited at a distribution company such as ATCC; the reagents themselves may ultimately be available through a licensed vendor. As for whether your lab can just make your own affinity clamp to your own special mark, Koide cautions: “The phage display that we use for the main directed evolution engine is very tricky. … This is not something the typical epigenetics lab would want to try.”