The 2018 Nobel Prize in Chemistry has gone to Frances Arnold (for directed evolution of enzymes) and to George Smith and Gregory Winter for phage display. These are worthy discoveries, techniques that have gone on to be used for a huge variety of work ranging from blue-sky research to marketed drugs, and the Nobel committee is definitely correct when they refer to them as having “harnessed the power of evolution”.
The proteins we have around us in living systems have, of course, been shaped by evolutionary forces. In fact, there’s really no better place to see the idea at work. On those occasions when I’ve encountered someone skeptical of the whole idea of evolution, I’ve wanted (and in some cases tried to) bring them up to speed on protein sequences and what they tell us. In short, that’s a history of random mutations, each of them put to the test in every organism. Fitness to reproduce asks several questions, very insistently, of any new mutation: does your current form help to pass on this new sequence? Do you at least not hurt the current chances of doing so? By chance, would you happen to be useful for anything else? Looking over related organisms at the protein-sequence and DNA-sequence level shows you a lurching, staggering mutational history as various residues get changed easily (to apparently not much effect), while others are strongly conserved (can’t touch ’em without trouble) and others lead to the new protein wandering into completely new roles over time.
Frances Arnold tried to harness this sort of thing deliberately. Here’s the 1993 paper on those efforts, which showed how a well-known enzyme (subtilisin) could be modified, by rounds of mutation and selection, to produce a variation that could still function in a decidedly non-natural environment (60% dimethylformamide/water, a brew that would stop most native proteins in their tracks). The abstract of the paper finished up by saying “Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures–it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo“, and that about sums it up. We don’t have to play the hand we’re dealt; we can shuffle the cards and try for something else, then keep the ones we like and draw to those to improve. (It has just occurred to me that draw poker is an excellent metaphor for the whole process!) There are a *lot* of protein features that we might want to engineer in, and this paper and its follow-ups have led to a whole new field that spends its time doing just that.
I wrote about Arnold’s most recent work just earlier this year, and over the years I’ve also written about enzymes engineered to fluorinate compounds, to do metal-catalyzed transformations, and to produce pharmaceutical intermediates to order. There’s still a great deal of chance and luck involved in these studies, but that’s not a complaint: chance and luck are what got us the proteins that are keeping us alive right now. The molecular machinery of biology lets you industrialize that sort of thing, running through huge numbers of variations and selecting out the interesting ones, in ways that regular organic chemistry is just not equipped to do. This area of research has come a long way in 25 years, but the horizon is still nowhere in sight.
The second half of today’s prize is for phage display, a technique that’s closely related to directed evolution of proteins. Bacteriophages are viruses that infect bacteria, and they display various peptides on their surfaces. George Smith’s breakthrough was to engineer one of these (PIII, previously not exactly a star player) to express new sequences. The idea was that these would show up on the phage’s surface, and that after it had had a chance to reproduce itself in some unlucky bacteria, you could take the resulting solution and flow it across some sort of affinity purification. That’s a basic and powerful technique in chemical and molecular biology: you have some sort of solid support (a column of resin, say) that has on it a known binding partner for your species of interest. You pour some sort of gemisch across this, and the species in it that stick the tightest to the resin binding partner stay there while you wash everything else off. You then switch to more vigorous solvent/buffer conditions to get the strong-binding things to wash off, and voila: you have purified out the best binders.
Smith did that with the phages in the mid-to-late 1980s – after all, they have proteins on their surface – and showed that such a protein-displaying phage would indeed be captured by affinity purification. As the technique was refined, he and his lab demonstrated that they could enhance the concentration of some particular phage by huge amounts in affinity purification (hundred-million-fold!) and this led to the next stage of things: setting up the phages so that they didn’t just display one particular protein that you stuck in there, but a whole range of variations, all produced randomly at the same time (those tools of molecular biology again). That had immediate applications in antibody work, since the whole thing about antibodies is that they recognize peptide sequences/surfaces with high affinity. Phage display suddenly made it possible to determine just exactly what sequences bound with the most affinity, and to produce comprehensive maps of what any particular antibody liked the most.
On the flip side, it also let you optimize synthetic antibody sequences to a given protein as well, and that was where Gregory Winter’s lab came in, showing that functional antibody sequences could be expressed on the phage surface. You can let this rip with millions upon millions of possibilities simultaneously, picking out the best binders from the whole collection. Then you can use the sequence that you found to design another round of more focused variations and send it back around again, etc. All this started getting reduced to practice (in both directions) in the early 1990s, and the worth of these techniques was utterly, obviously apparent at the very start. As were the sorts of variations you would want to try (different regions of the antibody protein, longer stretches of them, different gene-shuffling techniques to make variation libraries, optimizing antibodies against things that you couldn’t get by classic immunization techniques, and on and very much on).
These advances are what has led to the modern antibody industry. That ranges from sheer-research lab reagents, through diagnostic kits, all the way to multibillion dollar marketed therapies. Being able to run through gigantic piles of possibilities and enrich the best ones by factors of ten-to-the-whatever with every round of selection really opened up the field, as well it should have. There have been many advances over the last 25 years in making human-type antibodies directly, in different sorts of phages (and in using things for protein display beyond phage as well, such as yeast cells and whole bacteria), new affinity-selection techniques. . .a person could go on for quite a while, and I have to make a living (although, to be fair, these topics are directly relevant to how I make that living!) I can recommend the Nobel committee’s scientific background paper for more details and for leading references. If you’d like to see one way that all the topics of today’s award intersect, here’s a blog I wrote about a technique to use phage display to try to make new evolved enzymes – you can mix and match this stuff forever.
Or at least for a very long time. And that’s what’s going on now – the possibilities are very far from being exhausted, and the rewards are still huge. Protein engineering in general is going to be with us for a very long time to come, and I wouldn’t even want to guess what forms it might take. Congratulations to today’s winners! (And I can’t resist linking to Arnold’s Twitter account from a few days ago!)
Update: for those who have asked, I have some broader thoughts on the Chemistry Nobels and the Nobel prize awards in general, but those will have to wait until tomorrow. . .